Process for the biosynthesis of an ergot alkaloid derivative and use thereof
By employing synergistic fermentation of ergot and Bacillus subtilis, eutectic solvent extraction, and molecular imprinting purification techniques, the problems of cumbersome procedures and low yield in the production of ergot alkaloid derivatives have been solved, enabling efficient and green large-scale production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- 宝利化(南京)制药有限公司
- Filing Date
- 2025-06-24
- Publication Date
- 2026-05-19
AI Technical Summary
Existing methods for producing ergot alkaloid derivatives are cumbersome, have low yields, require harsh reaction conditions, produce complex and difficult-to-separate products, are greatly affected by the environment, and lack enantioselectivity, making it difficult to achieve green and efficient production.
The fermentation of ergot and Bacillus subtilis was carried out in a co-fermentation process, combined with eutectic solvent and supercritical CO2 extraction technology, and molecular imprinting purification. The fermentation conditions and extraction and purification process were optimized by regulating specific precursors-inducers and metal ions.
It significantly improved the yield and purity of ergot alkaloid derivatives, shortened the fermentation cycle, reduced production costs, and enabled green, efficient, and large-scale production.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biosynthesis technology, specifically to a biosynthesis process for ergot alkaloid derivatives and its application. Background Technology
[0002] Ergot alkaloids are a class of compounds with important pharmacological activities. Their unique tetracyclic indole structure enables them to interact with various neurotransmitter receptors, playing a crucial role in the treatment of neurological and vascular diseases. For example, dihydroergot alkaloid mesylate can be used to improve age-related cognitive impairment, sequelae of cerebrovascular diseases, vascular dementia, and migraines. However, current methods for producing ergot alkaloids still have many limitations, and there is an urgent need to develop more efficient, green, and sustainable preparation processes.
[0003] Traditional chemical synthesis methods typically involve multiple steps, which are cumbersome and result in low overall yields. Many critical reactions must be carried out under high temperature, high pressure, or highly corrosive reagents (such as concentrated sulfuric acid and organometallic catalysts), increasing energy consumption, operational risks, and environmental burden. Furthermore, chemical synthesis often struggles to precisely control stereoselectivity, leading to the existence of multiple isomers in the product, which complicates subsequent separation and purification, thus increasing production costs. Although advancements in asymmetric catalysis technologies in recent years (such as chiral ligands and transition metal catalysis) have improved this issue to some extent, the overall process still faces challenges in terms of economics and scalability.
[0004] Natural extraction methods mainly rely on ergot ( Claviceps purpurea Ergot alkaloid derivatives can be obtained by fermenting or field-infecting rye, but this method has the following problems:
[0005] (1) Complex products: Ergot metabolites are diverse, and target components (such as ergotamine, ergotamine, and ergotamine) often coexist with structural analogs, making separation and purification difficult and resulting in low recovery rates; (2) Environmentally sensitive: The growth of strains and the synthesis of secondary metabolites are easily affected by culture conditions such as temperature, pH, and dissolved oxygen, leading to significant batch-to-batch yield fluctuations; (3) Long production cycle: From strain cultivation to fermentation, extraction, and purification, the entire process takes several weeks or even months, resulting in low efficiency; (4) Ecological impact: Large-scale field planting may introduce the risk of Ergot contamination, while improper treatment of industrial fermentation wastewater may cause environmental pressure. Based on the above statements, this application provides a biosynthesis process for ergotamine derivatives and its application. Summary of the Invention
[0006] To address the shortcomings of existing technologies in the production of ergot alkaloid derivatives, such as cumbersome steps, low yield, harsh reaction conditions, complex and difficult-to-separate products, significant environmental influence, and lack of enantioselectivity, and to achieve green and efficient production of ergot alkaloid derivatives, this application provides a biosynthetic process for ergot alkaloid derivatives and its application.
[0007] In a first aspect, this application provides a biosynthetic process for ergot alkaloid derivatives, employing the following technical solution:
[0008] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0009] S1. Mix ergot and Bacillus subtilis and inoculate into fermentation medium for preliminary fermentation culture to obtain preliminary fermentation broth;
[0010] S2. Add the precursor-inducer complex system and metal ions to the initial fermentation broth for secondary fermentation culture to obtain the fermentation broth;
[0011] S3. Primary extraction of the fermentation broth is performed using a eutectic solvent, and the eutectic solvent phase is collected.
[0012] S4. The crude extract was obtained by refining the eutectic solvent phase using supercritical CO2 extraction technology.
[0013] S5. The crude extract was purified using molecular imprinting technology to obtain ergot alkaloid derivatives.
[0014] Preferably, in step S1, the live bacteria ratio of ergot to Bacillus subtilis is 9-11:1, and the total live bacteria count in the fermentation medium is 10. 6 -10 8 CFU / mL.
[0015] Preferably, the fermentation culture medium in step S1 is as follows: 40-60g rye flour, 16-24g mannitol, 4-6g yeast extract, 2.4-3.6g corn steep liquor, diluted with distilled water to 1L, sterilized at 121℃ and 0.1MPa for 15-25min, and the pH adjusted to 5.5-5.8.
[0016] Preferably, the preliminary fermentation parameters in step S1 are as follows: temperature 24-26℃, pH 5.5-5.8, aeration rate 0.8-1.4 vvm, stirring rate 400-600 rpm, dissolved oxygen 20-30%, and fermentation time 46-50 h.
[0017] Preferably, in step S2, the precursor-inducer complex system is L-tryptophan and ginkgo biloba extract; the metal ion is Zn. 2+ and Fe 3+It is added in the form of zinc sulfate and ferric sulfate.
[0018] Preferably, the precursor-inducer complex system and the method of adding metal ions in step S2 are as follows: L-tryptophan and ginkgo leaf extract are mixed at a mass ratio of 1:1, and a mother liquor of 8-12% (w / v) is prepared with sterile water. After filtration and sterilization, the mother liquor is added to the preliminary fermentation broth to a final concentration of 0.2-0.4% (w / v). Then, 0.05-0.1 mM sterile zinc sulfate and 0.04-0.06 mM sterile ferric sulfate are added.
[0019] Preferably, the secondary fermentation culture parameters in step S2 are as follows: temperature 24-26℃, pH 5.8-6.5, aeration rate 0.8-1.4 vvm, stirring rate 200-400 rpm, dissolved oxygen 5-15%, and fermentation time 48-72 h.
[0020] Preferably, the eutectic solvent in step S3 is obtained by mixing choline chloride, oxalic acid and water in a mass ratio of 0.5-1.5:1-2:0.1.
[0021] Preferably, the specific operation steps of the primary extraction in step S3 are as follows: centrifuge the fermentation broth at 6000-10000 rpm for 8-12 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, stir and extract for 2-3 h at 40-50℃ and 180-220 rpm, then centrifuge at 6000-10000 rpm for 10-20 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0022] Preferably, the specific operation steps of step S4 are as follows: the eutectic solvent phase is transferred to the extraction vessel of the supercritical extraction device, the extraction temperature is set to 30-35℃ and the pressure is 30-50MPa, ethanol is added to the eutectic solvent phase at a ratio of 5-8% (v / v), CO2 is introduced, and the extraction is circulated for 2-4 hours. The extract is collected, and CO2 and ethanol are removed by vacuum distillation to obtain the crude extract.
[0023] Preferably, the specific operation steps of step S5 are as follows: MIP resin with ergocinine as a template is soaked in methanol for 20-28 hours, washed with deionized water until neutral, and set aside; the crude extract is dissolved in phosphate buffer at pH 6.6-7.0 to prepare a loading solution with a concentration of 9-11 mg / mL; the loading solution is passed through a chromatography column packed with MIP resin at a flow rate of 0.5-1.5 mL / min for dynamic adsorption; after adsorption, the chromatography column is washed with phosphate buffer at pH 6.6-7.0 until the eluent is colorless; finally, elution is performed with a chloroform solution containing 4-6% (v / v) triethylamine, the eluent is collected, and concentrated under reduced pressure until the chloroform residue is less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0024] Secondly, this application provides an ergot alkaloid derivative prepared using the above-mentioned biosynthetic process of ergot alkaloid derivatives.
[0025] Thirdly, this application provides the use of the above-mentioned ergot alkaloid derivatives in the preparation of dihydroergot alkaloid mesylate-related drugs.
[0026] In summary, this application has the following beneficial effects:
[0027] (1) By using a specific ratio of ergot and Bacillus subtilis for synergistic fermentation, the metabolic complementarity and synergistic effect of the two strains is fully utilized. First, when the two strains are inoculated at the ratio of 9-11:1, the number of Bacillus subtilis can fully meet the cofactor supply required for the growth of ergot without excessively competing for nutrients in the culture medium. Second, at this ratio, the metabolites secreted by Bacillus subtilis can maintain the redox potential in the fermentation system within the optimal range of -320mV to -280mV, creating an ideal environment for the synthesis of ergot alkaloid derivatives. This optimized strain ratio increases the total yield of ergot alkaloid derivatives compared to fermentation with a single strain, while shortening the fermentation cycle.
[0028] (2) The phased dissolved oxygen control strategy can significantly improve the fermentation efficiency of ergot. Maintaining a high dissolved oxygen level in the initial fermentation stage can meet the oxygen demand for rapid cell growth and promote rapid biomass accumulation. After entering the secondary fermentation stage, the dissolved oxygen level is adjusted to a low dissolved oxygen state, which can effectively simulate the microaerobic conditions of ergot in its natural parasitic environment, trigger its defensive secondary metabolic response, significantly activate the expression of key enzymes in the ergot alkaloid synthesis pathway, increase ergot alkaloid production, and shorten the fermentation cycle.
[0029] (3) The addition of L-tryptophan and Ginkgo biloba extract has a synergistic effect. L-tryptophan, as a direct precursor of ergot alkaloid biosynthesis, provides a sufficient carbon skeleton source for the metabolic pathway; while Ginkgo biloba extract, through its unique flavonoids and terpene lactones, on the one hand, simulates the biological stress environment of the host plant, activates the secondary metabolic defense mechanism of ergot, and significantly upregulates the expression level of key enzyme genes in the tryptophan-ergot alkaloid synthesis pathway. On the other hand, it promotes the transmembrane transport of precursor substances by improving cell membrane permeability. At the same time, its powerful antioxidant components can effectively alleviate oxidative stress damage and prolong the stable alkaloid production period of the bacteria.
[0030] (4) Adding metal ions Fe 3+ and Zn 2+ It can significantly improve the efficiency of peptide cyclization. First, Fe 3+ As a redox cofactor, it can activate the active site of cyclases and promote the formation of intramolecular disulfide bonds; secondly, Zn 2+ By coordinating with the polypeptide backbone, the transition state conformation is stabilized, thereby reducing the activation energy of the cyclization reaction.
[0031] (5) The combination of choline chloride-oxalic acid-water eutectic solvent and supercritical CO2 extraction technology not only improves the extraction efficiency compared with traditional organic solvent extraction methods, but also effectively preserves bioactive components. The molecular imprinting purification step uses ergocinine as a template and is combined with a weakly basic chloroform elution system, which effectively improves the purity of the final product and avoids the destruction of the product by strong acid and strong base conditions.
[0032] (6) The entire process parameters have been rigorously optimized, exhibiting excellent repeatability and scalability. The culture medium uses inexpensive and readily available raw materials such as rye flour and mannitol, combined with standardized fermentation control conditions, which reduces production costs compared to traditional methods. The final product can be directly used as an important raw material for the preparation of drugs such as dihydroergot methyl methacrylate, providing a reliable technical solution for the large-scale production of ergot alkaloids. Detailed Implementation
[0033] The present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention.
[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.
[0036] The preparation steps of MIP resin using ergocinine as a template are as follows:
[0037] (1) Pre-assembly: Dissolve 0.5 mmol of ergocornine in 20 mL of methanol, add methacrylic acid (MAA), the molar ratio of MAA to ergocornine is 4:1, stir at 25℃ for 2 h to form a complex;
[0038] (2) Polymerization: Ethylene glycol dimethacrylate (EGDMA) was added to the composite, with a molar ratio of EGDMA to MAA of 20:1. Then, azobisisobutyronitrile (AIBN) of 1% by mass of MAA was added. Nitrogen gas was passed through to remove oxygen, and the polymer was thermally polymerized at 70°C for 24 h to obtain the polymer.
[0039] (3) Template elution: The polymer was ground and then extracted with methanol-acetic acid (9:1, v / v) by Soxhlet for 48 h until no ergocinine residue was detected by HPLC.
[0040] (4) Post-treatment: Particles with a size in the range of 25-50μm are sieved out and vacuum dried to obtain MIP resin with ergoconine as template.
[0041] Ginkgo biloba extract was purchased from Zhejiang Delekang Food Co., Ltd., CAS No.: 90045-36-6.
[0042] Ginsenosides were purchased from Chengdu Weiying Synthetic Biotechnology Co., Ltd., CAS No.: 14197-60-5.
[0043] Examples 1-3 provide a biosynthetic process for ergot alkaloid derivatives.
[0044] Example 1
[0045] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0046] S1. Mix ergot (accession number ATCC 26245) and Bacillus subtilis (accession number ATCC 6051) at a live bacteria ratio of 9:1, with a total live bacteria count of 10. 6 CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 40g rye flour, 16g mannitol, 4g yeast extract, and 2.4g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 15 minutes, and the pH was adjusted to 5.5. The initial fermentation conditions were set as follows: temperature 24℃, pH 5.5, aeration rate 0.8 vvm, stirring rate 400 rpm, dissolved oxygen 20%. The preliminary fermentation broth was obtained after 46 hours of fermentation.
[0047] S2. L-tryptophan and ginkgo leaf extract were mixed at a mass ratio of 1:1 and prepared into an 8% (w / v) stock solution with sterile water. After filtration and sterilization, the stock solution was added to the initial fermentation broth to achieve a final concentration of 0.2% (w / v). Then, 0.05 mM sterile zinc sulfate and 0.04 mM sterile ferric sulfate were added. The secondary fermentation was carried out at a temperature of 24℃, a pH of 5.8, an aeration rate of 0.8 vvm, a stirring rate of 200 rpm, and a dissolved oxygen level of 5%. The fermentation broth was obtained after 48 hours of fermentation.
[0048] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 0.5:1:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 6000 rpm for 8 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 40℃ and 180 rpm for 2 h. Then centrifuge at 6000 rpm for 10 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0049] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 30℃ and the pressure to 30MPa, add ethanol to the eutectic solvent phase at a ratio of 5% (v / v), introduce CO2, circulate and extract for 2 hours, collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0050] S5. MIP resin with ergocinine as template was soaked in methanol for 20 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 6.6 to prepare a loading solution with a concentration of 9 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 0.5 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 6.6 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 4% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0051] Example 2
[0052] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0053] S1. Mix ergot (accession number ATCC 26245) and Bacillus subtilis (accession number ATCC 6051) at a live bacteria ratio of 10:1, with a total live bacteria count of 10. 7CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 50g rye flour, 20g mannitol, 5g yeast extract, and 3g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 20 minutes, and the pH was adjusted to 5.6. The preliminary fermentation conditions were set as follows: temperature 25℃, pH 5.6, aeration rate 1.1 vvm, stirring rate 500 rpm, dissolved oxygen 25%. The preliminary fermentation broth was obtained after 48 hours of fermentation.
[0054] S2. L-tryptophan and ginkgo leaf extract were mixed at a mass ratio of 1:1 and prepared into a 10% (w / v) stock solution with sterile water. After filtration and sterilization, the stock solution was added to the initial fermentation broth to achieve a final concentration of 0.3% (w / v). Then, 0.075 mM sterile zinc sulfate and 0.05 mM sterile ferric sulfate were added. The secondary fermentation was set at a temperature of 25°C, a pH of 6.2, an aeration rate of 1.1 vvm, a stirring rate of 300 rpm, and a dissolved oxygen level of 10%. The fermentation broth was obtained after 60 hours of fermentation.
[0055] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 1:1.5:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 45℃ and 200 rpm for 2.5 h. Then centrifuge at 8000 rpm for 15 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0056] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 32℃ and the pressure to 40MPa, add ethanol to the eutectic solvent phase at a ratio of 6.5% (v / v), introduce CO2, and circulate for extraction for 3 hours. Collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0057] S5. MIP resin with ergocinine as template was soaked in methanol for 24 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 6.8 to prepare a loading solution with a concentration of 10 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 1 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 6.8 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 5% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0058] Example 3
[0059] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0060] S1. Mix ergot (accession number ATCC 26245) and Bacillus subtilis (accession number ATCC 6051) at a live bacteria ratio of 11:1, with a total live bacteria count of 10. 8 CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 60g rye flour, 24g mannitol, 6g yeast extract, and 3.6g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 25 minutes, and the pH was adjusted to 5.8. The initial fermentation conditions were set as follows: temperature 26℃, pH 5.8, aeration rate 1.4 vvm, stirring rate 600 rpm, dissolved oxygen 30%. The preliminary fermentation broth was obtained after 50 hours of fermentation.
[0061] S2. L-tryptophan and ginkgo leaf extract were mixed at a mass ratio of 1:1 and prepared into a 12% (w / v) stock solution with sterile water. After filtration and sterilization, the stock solution was added to the initial fermentation broth to achieve a final concentration of 0.4% (w / v). Then, 0.1 mM sterile zinc sulfate and 0.06 mM sterile ferric sulfate were added. The secondary fermentation was carried out at a temperature of 26℃, a pH of 6.5, an aeration rate of 1.4 vvm, a stirring rate of 400 rpm, and a dissolved oxygen level of 15%. The fermentation broth was obtained after 72 hours of fermentation.
[0062] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 1.5:2:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 10000 rpm for 12 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 50℃ and 220 rpm for 3 h. Then centrifuge at 10000 rpm for 20 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0063] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 35℃ and the pressure to 50MPa, add ethanol to the eutectic solvent phase at a ratio of 8% (v / v), introduce CO2, circulate and extract for 4 hours, collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0064] S5. MIP resin with ergocinine as template was soaked in methanol for 28 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 7.0 to prepare a loading solution with a concentration of 11 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 1.5 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 7.0 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 6% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0065] Comparative Example 1
[0066] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0067] S1. Ergot (accession number ATCC 26245) was prepared with a total viable count of 10... 7 CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 50g rye flour, 20g mannitol, 5g yeast extract, and 3g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 20 minutes, and the pH was adjusted to 5.6. The preliminary fermentation conditions were set as follows: temperature 25℃, pH 5.6, aeration rate 1.1 vvm, stirring rate 500 rpm, dissolved oxygen 25%. The preliminary fermentation broth was obtained after 48 hours of fermentation.
[0068] S2. L-tryptophan and ginkgo leaf extract were mixed at a mass ratio of 1:1 and prepared into a 10% (w / v) stock solution with sterile water. After filtration and sterilization, the stock solution was added to the initial fermentation broth to achieve a final concentration of 0.3% (w / v). Then, 0.075 mM sterile zinc sulfate and 0.05 mM sterile ferric sulfate were added. The secondary fermentation was set at a temperature of 25°C, a pH of 6.2, an aeration rate of 1.1 vvm, a stirring rate of 300 rpm, and a dissolved oxygen level of 10%. The fermentation broth was obtained after 60 hours of fermentation.
[0069] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 1:1.5:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 45℃ and 200 rpm for 2.5 h. Then centrifuge at 8000 rpm for 15 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0070] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 32℃ and the pressure to 40MPa, add ethanol to the eutectic solvent phase at a ratio of 6.5% (v / v), introduce CO2, and circulate for extraction for 3 hours. Collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0071] S5. MIP resin with ergocinine as template was soaked in methanol for 24 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 6.8 to prepare a loading solution with a concentration of 10 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 1 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 6.8 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 5% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0072] Comparative Example 2
[0073] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0074] S1. Mix *Ergotae* (accession number ATCC 26245) and *Bacillus licheniformis* (accession number ATCC 14580) at a live bacteria ratio of 10:1, with a total live bacteria count of 10... 7 CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 50g rye flour, 20g mannitol, 5g yeast extract, and 3g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 20 minutes, and the pH was adjusted to 5.6. The preliminary fermentation conditions were set as follows: temperature 25℃, pH 5.6, aeration rate 1.1 vvm, stirring rate 500 rpm, dissolved oxygen 25%. The preliminary fermentation broth was obtained after 48 hours of fermentation.
[0075] S2. L-tryptophan and ginkgo leaf extract were mixed at a mass ratio of 1:1 and prepared into a 10% (w / v) stock solution with sterile water. After filtration and sterilization, the stock solution was added to the initial fermentation broth to achieve a final concentration of 0.3% (w / v). Then, 0.075 mM sterile zinc sulfate and 0.05 mM sterile ferric sulfate were added. The secondary fermentation was set at a temperature of 25℃, a pH of 6.2, an aeration rate of 1.1 vvm, a stirring rate of 300 rpm, and a dissolved oxygen level of 10%. The fermentation broth was obtained after 60 hours of fermentation.
[0076] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 1:1.5:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 45℃ and 200 rpm for 2.5 h. Then centrifuge at 8000 rpm for 15 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0077] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 32℃ and the pressure to 40MPa, add ethanol to the eutectic solvent phase at a ratio of 6.5% (v / v), introduce CO2, and circulate for extraction for 3 hours. Collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0078] S5. MIP resin with ergocinine as template was soaked in methanol for 24 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 6.8 to prepare a loading solution with a concentration of 10 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 1 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 6.8 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 5% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0079] Comparative Example 3
[0080] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0081] S1. Mix ergot (accession number ATCC 26245) and Bacillus subtilis (accession number ATCC 6051) at a live bacteria ratio of 10:1, with a total live bacteria count of 10. 7 CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 50g rye flour, 20g mannitol, 5g yeast extract, and 3g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 20 minutes, and the pH was adjusted to 5.6. The preliminary fermentation conditions were set as follows: temperature 25℃, pH 5.6, aeration rate 1.1 vvm, stirring rate 500 rpm, dissolved oxygen 25%. The preliminary fermentation broth was obtained after 48 hours of fermentation.
[0082] S2. Prepare a 10% (w / v) stock solution of L-tryptophan with sterile water. After filtration and sterilization, add it to the initial fermentation broth to achieve a final concentration of 0.3% (w / v). Then add 0.075 mM sterile zinc sulfate and 0.05 mM sterile ferric sulfate. Set the temperature for the secondary fermentation to 25℃, pH to 6.2, aeration rate to 1.1 vvm, stirring rate to 300 rpm, and dissolved oxygen to 10%. After 60 hours of fermentation, the fermentation broth is obtained.
[0083] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 1:1.5:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 45℃ and 200 rpm for 2.5 h. Then centrifuge at 8000 rpm for 15 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0084] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 32℃ and the pressure to 40MPa, add ethanol to the eutectic solvent phase at a ratio of 6.5% (v / v), introduce CO2, and circulate for extraction for 3 hours. Collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0085] S5. MIP resin with ergocinine as template was soaked in methanol for 24 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 6.8 to prepare a loading solution with a concentration of 10 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 1 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 6.8 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 5% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0086] Comparative Example 4
[0087] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0088] S1. Mix ergot (accession number ATCC 26245) and Bacillus subtilis (accession number ATCC 6051) at a live bacteria ratio of 10:1, with a total live bacteria count of 10. 7CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 50g rye flour, 20g mannitol, 5g yeast extract, and 3g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 20 minutes, and the pH was adjusted to 5.6. The preliminary fermentation conditions were set as follows: temperature 25℃, pH 5.6, aeration rate 1.1 vvm, stirring rate 500 rpm, dissolved oxygen 25%. The preliminary fermentation broth was obtained after 48 hours of fermentation.
[0089] S2. Mix L-tryptophan and ginsenosides at a mass ratio of 1:1, prepare a 10% (w / v) stock solution with sterile water, filter to remove bacteria, and add it to the initial fermentation broth to achieve a final concentration of 0.3% (w / v). Then add 0.075 mM sterile zinc sulfate and 0.05 mM sterile ferric sulfate. Set the temperature for the secondary fermentation to 25℃, pH to 6.2, aeration rate to 1.1 vvm, stirring rate to 300 rpm, and dissolved oxygen to 10%. After fermentation for 60 hours, the fermentation broth is obtained.
[0090] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 1:1.5:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 45℃ and 200 rpm for 2.5 h. Then centrifuge at 8000 rpm for 15 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0091] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 32℃ and the pressure to 40MPa, add ethanol to the eutectic solvent phase at a ratio of 6.5% (v / v), introduce CO2, and circulate for extraction for 3 hours. Collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0092] S5. MIP resin with ergocinine as template was soaked in methanol for 24 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 6.8 to prepare a loading solution with a concentration of 10 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 1 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 6.8 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 5% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0093] Comparative Example 5
[0094] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0095] S1. Mix ergot (accession number ATCC 26245) and Bacillus subtilis (accession number ATCC 6051) at a live bacteria ratio of 10:1, with a total live bacteria count of 10. 7 CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 50g rye flour, 20g mannitol, 5g yeast extract, and 3g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 20 minutes, and the pH was adjusted to 5.6. The preliminary fermentation conditions were set as follows: temperature 25℃, pH 5.6, aeration rate 1.1 vvm, stirring rate 500 rpm, dissolved oxygen 25%. The preliminary fermentation broth was obtained after 48 hours of fermentation.
[0096] S2. L-tryptophan and ginkgo leaf extract were mixed at a mass ratio of 1:1 and prepared into a 10% (w / v) stock solution with sterile water. After filtration and sterilization, the stock solution was added to the initial fermentation broth to achieve a final concentration of 0.3% (w / v). Then, 0.075 mM sterile zinc sulfate and 0.05 mM sterile ferric sulfate were added. The secondary fermentation was set at a temperature of 25°C, a pH of 6.2, an aeration rate of 1.1 vvm, a stirring rate of 300 rpm, and a dissolved oxygen level of 10%. The fermentation broth was obtained after 60 hours of fermentation.
[0097] S3. Mix chloroform and methanol at a ratio of 2:1 (v / v) to obtain a chloroform-methanol mixed solvent. Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the cells, collect the supernatant, add an equal volume of chloroform-methanol mixed solvent to the supernatant, and extract by stirring at 45℃ and 200 rpm for 2.5 h. Then centrifuge at 8000 rpm for 15 min to separate the organic phase and the aqueous phase, and collect the organic phase.
[0098] S4. Transfer the organic phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 32℃ and the pressure to 40MPa, add ethanol to the eutectic solvent phase at a ratio of 6.5% (v / v), introduce CO2, and circulate for extraction for 3 hours. Collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0099] S5. MIP resin with ergocinine as template was soaked in methanol for 24 h and washed with deionized water until neutral. The crude extract was dissolved in phosphate buffer at pH 6.8 to prepare a loading solution with a concentration of 10 mg / mL. The loading solution was passed through a chromatography column packed with MIP resin at a flow rate of 1 mL / min for dynamic adsorption. After adsorption, the chromatography column was washed with phosphate buffer at pH 6.8 until the eluent was colorless. Finally, the column was eluted with chloroform solution containing 5% (v / v) triethylamine. The eluent was collected and concentrated under reduced pressure until the chloroform residue was less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
[0100] Comparative Example 6
[0101] A biosynthetic process for an ergot alkaloid derivative includes the following steps:
[0102] S1. Mix ergot (accession number ATCC 26245) and Bacillus subtilis (accession number ATCC 6051) at a live bacteria ratio of 10:1, with a total live bacteria count of 10. 7 CFU / mL was inoculated into the fermentation medium for preliminary fermentation. The fermentation medium consisted of 50g rye flour, 20g mannitol, 5g yeast extract, and 3g corn steep liquor, with distilled water added to a final volume of 1L. The mixture was sterilized at 121℃ and 0.1 MPa for 20 minutes, and the pH was adjusted to 5.6. The preliminary fermentation conditions were set as follows: temperature 25℃, pH 5.6, aeration rate 1.1 vvm, stirring rate 500 rpm, dissolved oxygen 25%. The preliminary fermentation broth was obtained after 48 hours of fermentation.
[0103] S2. L-tryptophan and ginkgo leaf extract were mixed at a mass ratio of 1:1 and prepared into a 10% (w / v) stock solution with sterile water. After filtration and sterilization, the stock solution was added to the initial fermentation broth to achieve a final concentration of 0.3% (w / v). Then, 0.075 mM sterile zinc sulfate and 0.05 mM sterile ferric sulfate were added. The secondary fermentation was set at a temperature of 25°C, a pH of 6.2, an aeration rate of 1.1 vvm, a stirring rate of 300 rpm, and a dissolved oxygen level of 10%. The fermentation broth was obtained after 60 hours of fermentation.
[0104] S3. Weigh out choline chloride, oxalic acid and water in a mass ratio of 1:1.5:0.1, mix them evenly, and place them in a 40℃ water bath to stir and dissolve them to form a eutectic solvent. Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the cells, collect the supernatant, add an equal volume of eutectic solvent to the supernatant, and extract by stirring at 45℃ and 200 rpm for 2.5 h. Then centrifuge at 8000 rpm for 15 min to separate the eutectic solvent phase and the aqueous phase, and collect the eutectic solvent phase.
[0105] S4. Transfer the eutectic solvent phase to the extraction vessel of the supercritical extraction device, set the extraction temperature to 32℃ and the pressure to 40MPa, add ethanol to the eutectic solvent phase at a ratio of 6.5% (v / v), introduce CO2, and circulate for extraction for 3 hours. Collect the extract, remove CO2 and ethanol by vacuum distillation to obtain the crude extract.
[0106] S5. The crude extract was dissolved in dichloromethane-methanol at a ratio of 9:1 (v / v) and then mixed with 100-mesh silica gel at a ratio of 1:10 (w / w). The sample was dried under reduced pressure to obtain a silica gel-loaded sample. The sample was loaded onto a silica gel column and then washed with petroleum ether-ethyl acetate at a ratio of 8:2 (v / v) to remove nonpolar impurities, followed by elution of the target product with petroleum ether-ethyl acetate at a ratio of 6:4 (v / v). Finally, polar impurities were removed with dichloromethane-methanol at a ratio of 9:1 (v / v). The eluent was collected and concentrated under reduced pressure until the residual amount of dichloromethane-methanol was less than 300 ppm as determined by GC analysis, thus obtaining the purified ergot alkaloid derivative.
[0107] Quantitative determination by high performance liquid chromatography
[0108] Quantitative analysis by high performance liquid chromatography (HPLC) was performed using a C18 reversed-phase column (4.6 × 250 mm, 5 μm). Acetonitrile was used as phase A, and ultrapure water containing 0.1% phosphoric acid was used as phase B. Gradient elution was performed, with the proportion of acetonitrile gradually increasing from 20% to 80% in the gradient elution. The flow rate was 1.0 mL / min, and the detection was performed at a wavelength of 280 nm. Standard curves were established using different ergot alkaloid derivative standards. The concentrations of each component were calculated based on the peak areas of the samples, and the total yield was obtained. The yields of Examples 1-3 and Comparative Examples 1-6 are shown in Table 1.
[0109] Table 1 Total production of ergot alkaloid derivatives
[0110]
[0111] As shown in Table 1 above, the total yield data of ergot alkaloid derivatives indicates that different process conditions significantly affect the yield. The total yield of Examples 1-3 ranged from 241.6 to 259.2 mg / L, with Example 2 achieving the highest yield of 259.2 mg / L. Comparative Example 1, with a single ergot fermentation yield of only 204.1 mg / L, demonstrates that the synergistic effect of Bacillus subtilis can increase the total yield of ergot alkaloid derivatives. In Comparative Example 2, replacing Bacillus subtilis with Bacillus licheniformis reduced the yield to 234.3 mg / L, indicating that the synergistic fermentation effect of Bacillus subtilis and ergot alkaloids was superior. The yields of Comparative Examples 3-4 were lower than those of Examples 1-3, highlighting that the synergistic effect of the L-tryptophan and Ginkgo biloba extract complex system is more conducive to the synthesis of ergot alkaloid derivatives. Comparative Examples 5 and 6, using conventional extraction and silica gel column purification respectively, had lower yields than Examples 1-3, demonstrating the advantages of eutectic solvent extraction and molecular imprinting technology in improving extraction efficiency and purification selectivity. In summary, the process described in this application significantly improves the yield of ergot alkaloid derivatives through multi-stage synergistic optimization, providing strong support for industrial production.
[0112] High performance liquid chromatography purity analysis
[0113] The purified product was prepared into a 1 mg / mL solution with methanol, filtered through a 0.22 μm filter membrane, and then subjected to gradient elution on a C18 reversed-phase column (4.6 × 250 mm, 5 μm) with acetonitrile as phase A and ultrapure water containing 0.1% phosphoric acid as phase B. The proportion of acetonitrile in the gradient elution was gradually increased from 20% to 80%, the flow rate was 1.0 mL / min, and the sample was detected at a wavelength of 280 nm. The chromatogram was recorded, and the percentage of the main peak area to the total peak area was calculated. The purity of Examples 1-3 and Comparative Examples 1-6 is shown in Table 2.
[0114] Table 2 Purity of Ergot alkaloid derivatives
[0115]
[0116] As shown in Table 2 above, the purity data of ergot alkaloid derivatives indicate that different process conditions significantly affect product purity. Examples 1-3 employed methods such as synergistic fermentation, the addition of a precursor-inducer complex system and metal ions for precursor-directed transformation, eutectic solvent extraction, supercritical CO2 extraction, and molecular imprinting, resulting in high product purity, with Example 2 exhibiting the highest purity. Comparative Examples 1-2, due to single-strain fermentation or replacement of synergistic bacteria, showed lower purity compared to Examples 1-3, indicating that the synergistic fermentation system can reduce the formation of other metabolites. Comparative Examples 3 and 4 showed lower purity than Examples 1-3, highlighting that the addition of a precursor-inducer complex system is more conducive to the directed synthesis of ergot alkaloid derivatives. Comparative Examples 5-6, using traditional solvent extraction and silica gel column chromatography respectively, showed significantly lower purity than the examples, highlighting the selective extraction of eutectic solvents and the precise identification advantages of molecular imprinting technology. In summary, the process described in this application significantly improves the purity of ergot alkaloid derivatives through methods such as synergistic fermentation, the addition of a precursor-inducer complex system and metal ions for precursor-directed transformation, eutectic solvent extraction, supercritical CO2 extraction, and molecular imprinting technology, thus providing a high-quality guarantee for drug preparation.
[0117] The above specific embodiments are merely explanations of this application and are not intended to limit this application. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A biosynthetic process for ergot alkaloid derivatives, characterized in that, Includes the following steps: S1. Mix ergot and Bacillus subtilis and inoculate into fermentation medium for preliminary fermentation culture to obtain preliminary fermentation broth; S2. Add the precursor-inducer complex system and metal ions to the initial fermentation broth for secondary fermentation culture to obtain the fermentation broth; S3. Primary extraction of the fermentation broth is performed using a eutectic solvent, and the eutectic solvent phase is collected. S4. The crude extract was obtained by refining the eutectic solvent phase using supercritical CO2 extraction technology. S5. The crude extract was purified using molecular imprinting technology to obtain ergot alkaloid derivatives. In step S1, the viable count ratio of Ergot cristatum to Bacillus subtilis is 9-11:1, and the total viable count in the fermentation medium is 10. 6 -10 8 CFU / mL; The fermentation medium in step S1 is as follows: 40-60g rye flour, 16-24g mannitol, 4-6g yeast extract, 2.4-3.6g corn steep liquor, add distilled water to a final volume of 1L, sterilize at 121℃ and 0.1 MPa for 15-25min, and adjust the pH to 5.5-5.
8. The preliminary fermentation parameters in step S1 are as follows: temperature 24-26℃, pH 5.5-5.8, aeration rate 0.8-1.4 vvm, stirring rate 400-600 rpm, dissolved oxygen 20-30%, and fermentation time 46-50 h. In step S2, the precursor-inducer complex system consists of L-tryptophan and ginkgo biloba extract; the metal ion is Zn. 2+ and Fe 3+ It is added in the form of zinc sulfate and ferric sulfate; In step S3, the eutectic solvent is obtained by mixing choline chloride, oxalic acid, and water in a mass ratio of 0.5-1.5:1-2:0.
1.
2. The biosynthesis process of ergot alkaloid derivatives according to claim 1, characterized in that, The specific operation steps of step S4 are as follows: the eutectic solvent phase is transferred to the extraction vessel of the supercritical extraction device, the extraction temperature is set to 30-35℃ and the pressure is 30-50MPa, ethanol is added to the eutectic solvent phase at a ratio of 5-8% (v / v), CO2 is introduced, and the extraction is circulated for 2-4 hours. The extract is collected, and CO2 and ethanol are removed by vacuum distillation to obtain the crude extract.
3. The biosynthesis process for ergot alkaloid derivatives according to claim 1, characterized in that, The specific steps of step S5 are as follows: MIP resin with ergocinine as a template is soaked in methanol for 20-28 hours, washed with deionized water until neutral, and set aside. The crude extract is dissolved in phosphate buffer at pH 6.6-7.0 to prepare a loading solution with a concentration of 9-11 mg / mL. The loading solution is passed through a chromatography column packed with MIP resin at a flow rate of 0.5-1.5 mL / min for dynamic adsorption. After adsorption, the chromatography column is washed with phosphate buffer at pH 6.6-7.0 until the eluent is colorless. Finally, elution is performed with a chloroform solution containing 4-6% (v / v) triethylamine. The eluent is collected and concentrated under reduced pressure until the chloroform residue is less than 60 ppm as determined by GC to obtain purified ergocinine derivatives.
4. An ergot alkaloid derivative prepared by the biosynthesis process of any one of claims 1-3.
5. The use of the ergot alkaloid derivative of claim 4 in the preparation of dihydroergot alkaloid mesylate-related drugs.