Application of echinacoside in preparation of medicine for improving autism social and cognitive impairment
By combining recombinant Saccharomyces cerevisiae strains with metabolic flux reprogramming fermentation, and utilizing autism-specific dual-ligand and pH-sensitive liposome technology, the problem of drug distribution in non-target areas was solved, achieving precise targeted drug delivery and efficient improvement of social and cognitive impairments in autism.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN CHILDRENS HOSPITAL
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-19
AI Technical Summary
Existing drug regimens for social and cognitive impairments in autism involve drugs that are widely distributed outside the target area, resulting in insufficient effective concentrations in the target area and making it difficult to specifically improve pathological damage.
By combining the construction of recombinant Saccharomyces cerevisiae strains with metabolic flux reprogramming fermentation, the targeted enrichment and directed release of echinacosides were achieved through affinity chromatography with dual ligands specific to the autism brain region and pH-sensitive liposome modification.
This achieved precise targeted drug delivery, increased the effective concentration in the target area, enhanced the targeted improvement of social and cognitive impairment in autistic patients, and improved the long-term storage stability of the formulation.
Smart Images
Figure CN122056903A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of medicinal chemistry, and more specifically, to the use of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients. Background Technology
[0002] Autism, a neurodevelopmental disorder, is characterized by core clinical manifestations including impairments in social interaction, cognitive function, and repetitive, stereotyped behaviors, severely affecting patients' social adaptability. With in-depth research into the pathological mechanisms of this disease, relevant technologies have clearly established that its pathogenesis is closely related to imbalances in neuroinflammation, synaptic dysfunction, excessive oxidative stress, and disruptions in neural circuits in key brain regions. Therefore, developing drugs that can specifically intervene in these pathological processes has become a key research focus in this field.
[0003] In current treatment plans for social and cognitive impairments in autism, most approaches combine conventional drug formulations with pan-targeted delivery systems. However, these methods fail to accurately identify specific targets in key brain regions associated with autism and rely solely on systemic drug distribution to attempt to exert their effects. This results in a large amount of drug being distributed in non-target areas, leading to insufficient effective concentrations in the target areas and making it difficult to specifically improve pathological damage. Summary of the Invention
[0004] To address the problem that existing treatments for social and cognitive impairment in autism involve drugs that are widely distributed outside the target area, resulting in insufficient effective concentrations in the target area and difficulty in specifically improving pathological damage, this application provides the application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autism.
[0005] The application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients, as provided in this application, adopts the following technical solution: The application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients includes the following steps: A recombinant Saccharomyces cerevisiae strain was constructed, precursor competitive pathway genes were knocked out, and a precursor synthesis enhancement module containing a constitutive promoter and an echinacoside synthesis module containing an inducible promoter were introduced to obtain the recombinant strain; The recombinant strain was subjected to metabolic flux reprogramming fermentation, with the pH adjusted in stages to 5.8–6.2 and 4.8–5.2, and 0.2%–0.6% methanol was added in a gradient to induce fermentation and obtain the fermentation broth. The fermentation broth was centrifuged at 7000-9000 r / min and pretreated with an 8-12 kDa ultrafiltration membrane to remove impurities, forming a pretreated solution. The pretreatment solution was purified by dual-target affinity chromatography, and echinacoside was obtained by gradient elution on a chromatography column coupled with a specific dual ligand for the autism brain region. Echinacea glycosides were prepared into liposomes containing pH-sensitive polyhistidine, which were then homogenized under high pressure at 600-1000 bar and modified with dual ligands to obtain a suspension. By adding a lyophilization protectant to the suspension and performing gradient pre-freezing and vacuum lyophilization, a drug to improve social and cognitive impairment in autistic patients was obtained.
[0006] By employing the above-mentioned technical solution, which combines the construction of recombinant Saccharomyces cerevisiae strains with metabolic flux reprogramming fermentation, echinacoside can be synthesized efficiently. Affinity chromatography coupled with a specific dual-ligand for autism brain regions enables targeted enrichment of echinacoside. It is then prepared into liposomes containing pH-sensitive polyhistidine and modified to allow the drug to accurately identify autism brain regions. Simultaneously, the pH-sensitive properties promote targeted drug release in the target area. Therefore, precise targeting and high efficacy are achieved, solving the problem in existing solutions for autism-related social and cognitive impairments where the drug is widely distributed in non-target areas, resulting in insufficient effective concentrations in the target area and difficulty in specifically improving pathological damage.
[0007] Preferably, the construction of the recombinant Saccharomyces cerevisiae strain includes the following steps: The TYR1 and GSY1 genes of Saccharomyces cerevisiae were knocked out using CRISPR-Cas9 technology to obtain knockout yeast; The precursor synthesis enhancement module and the echinacea glycoside synthesis module were co-transferred into the knockout strain. Positive strains were obtained through uracil auxotrophic screening to form a recombinant Saccharomyces cerevisiae strain. The precursor synthesis enhancement module contains the G6PDH gene and the PAL gene and is expressed using the TEF1 constitutive promoter. The echinacea glycoside synthesis module contains the UGT76G1 gene and the CYP73A5 gene and is expressed using the GAL1 inducible promoter.
[0008] By adopting the above technical solution, the targeted design of gene knockout and dual synthesis module works synergistically to block the non-targeted consumption of precursor substances. At the same time, by using the temporal regulation of different promoters, the accumulation of precursors and the synthesis of echinacoside are matched, thereby ensuring the efficient and stable synthesis of echinacoside and reducing interference from byproducts.
[0009] Preferably, the metabolic flux reprogramming fermentation of the recombinant strain includes the following steps: The seed culture of the recombinant Saccharomyces cerevisiae strain was inoculated into the fermenter at an inoculation rate of 8%~12% (v / v) and cultured at 30~32℃, 160~200 r / min and an aeration rate of 0.8~1.2 vvm. During the first 0-24 hours of cultivation, the pH was maintained at 5.8-6.2. After 24 hours, the pH was adjusted to 4.8-5.2. Methanol was added in three separate additions at 12-hour intervals, with each addition being 0.2%-0.6% (v / v). The fermentation cycle was 68-76 hours. The concentration of echinacoside in the fermentation broth was 200-400 mg / L, and the content of tyrosol, a byproduct, was ≤8 mg / L.
[0010] By adopting the above technical solution, the combination of parameters such as inoculum size, temperature, aeration rate, segmented pH, and gradient induction forms a synergistic effect, which not only provides a suitable environment for bacterial growth, but also activates the efficient expression of the synthesis module, thereby achieving high yield and low by-product of echinacoside.
[0011] Preferably, the centrifugation time is 8-12 min, the pressure of the ultrafiltration membrane pretreatment is 0.1-0.3 MPa, the removal rate of macromolecular impurities in the pretreatment solution is ≥95%, and the transmittance is ≥90%.
[0012] By adopting the above technical solution and matching the centrifugation time with the ultrafiltration pressure, it is possible to efficiently separate cell fragments and macromolecular impurities in the fermentation broth and obtain a high-purity pretreated solution.
[0013] Preferably, obtaining echinacea glycosides includes the following steps: The pretreatment solution was passed through a chromatography column containing a specific dual ligand for the autism brain region at a flow rate of 0.8–1.2 mL / min to bind echinacoside to the dual ligand. Impurities were eluted with Tris-HCl buffer containing 0.04–0.06 mol / L imidazole at pH 7.2–7.6, and then the bound echinacoside was eluted with Tris-HCl buffer containing 0.08–0.12 mol / L imidazole at pH 6.6–7.0. After elution, the echinacoside was purified by reversed-phase HPLC with methanol-water as the mobile phase (volume ratio 35:65~45:55) and a flow rate of 0.8~1.2 mL / min to obtain echinacoside with a purity ≥99.5%.
[0014] By adopting the above technical solution, the synergy of sample loading flow rate, gradient elution conditions and HPLC purification parameters can achieve the separation of echinacoside from impurities and proteins, improve product purity, reduce the loss of effective components, and ensure purification yield and product quality.
[0015] Preferably, the autism brain region-specific dual ligands are OXTR-Ab and 5-HTP, with the molar ratio of OXTR-Ab to the chromatography column carrier being 1:40 to 1:60, and the molar ratio of OXTR-Ab to 5-HTP being 1.5:1 to 2.5:1.
[0016] By adopting the above technical solution, the combination and ratio optimization of the two specific ligands form a synergistic targeting effect, which can identify the corresponding receptors in key brain regions of autism, enhance the targeted enrichment effect of affinity chromatography, and provide a specific basis for the targeted delivery of subsequent formulations.
[0017] Preferably, obtaining the suspension includes the following steps: PC, Chol and pH-sensitive polyhistidine in a mass ratio of 8~12:4~6:1~1.5 were added to anhydrous ethanol at 3~5 times their total mass and stirred in a water bath at 60~65℃ for 15~25 min to prepare liposome membrane material. Echinacoside was dissolved in physiological saline to prepare an aqueous solution of 8-12 mg / mL. Anhydrous ethanol is added to the liposome membrane material and dissolved by stirring in a water bath at 60-65°C to form an organic phase. Then, it is mixed with the aqueous phase solution at a volume ratio of 1:3 to 1:5 and stirred at 60-65°C for 25-35 minutes to form colostrum liposomes.
[0018] By adopting the above technical solution, the synergy of the membrane material ratio, the amount of anhydrous ethanol, and the organic-water phase ratio can form structurally stable colostrum liposomes, ensuring the efficient encapsulation of echinacoside.
[0019] Preferably, the modification of the dual ligands after homogenization under high pressure (600-1000 bar) includes the following steps: Colostrum liposomes were homogenized under high pressure of 600-1000 bar 4-6 times to form liposome suspensions; Mix OXTR-Ab and 5-HTP at a molar ratio of 1.5:1 to 2.5:1 until homogeneous, add liposome suspension, and incubate at 35 to 39°C for 1.5 to 2.5 hours to allow the dual ligands to be covalently linked to the liposome surface, forming a suspension.
[0020] By adopting the above technical solution, the synergy between high-pressure homogenization and ligand incubation conditions not only ensures the uniformity and stability of liposome particle size, but also promotes the strong binding of dual ligands to liposomes, thereby enhancing the targeted recognition capability of the formulation.
[0021] Preferably, the freeze-drying protectant is a trehalose-mannitol complex, and the amount added is 8% to 12% of the suspension mass, and the mass ratio of trehalose to mannitol is 2.5:1 to 3.5:1.
[0022] By adopting the above technical solution, the combination and ratio design of the two protective agents form a synergistic stabilizing effect, which can effectively protect the structural integrity of liposomes during freeze-drying and improve the long-term storage stability of the formulation.
[0023] Preferably, the gradient pre-freezing and vacuum freeze-drying includes the following steps: The suspension with added freeze-drying protectant was cooled to -8 to -12°C at a rate of 4 to 6°C / min and held at that temperature for 0.8 to 1.2 hours. Then it was cooled to -38 to -42°C at a rate of 8 to 12°C / min and held at that temperature for 1.5 to 2.5 hours to form a pre-frozen product. The pre-frozen material was dried under a vacuum of 8-12 Pa and a temperature of -22 to -18 °C for 10-14 hours, and then dried at a temperature of 23-27 °C for 3-5 hours.
[0024] By adopting the above technical solution, the synergy of gradient pre-freezing rate, constant temperature time and vacuum drying can smoothly complete the freeze-drying of the formulation, which not only preserves the drug activity and liposome structural stability, but also ensures the homogeneity of the formulation after reconstitution, thus guaranteeing the reliability of clinical use.
[0025] In summary, this application has the following beneficial effects: 1. Because this application uses a combination of recombinant Saccharomyces cerevisiae strain construction and metabolic flux reprogramming fermentation, it can efficiently synthesize echinacoside. By using affinity chromatography coupled with a specific dual ligand for autism brain regions, echinacoside is targeted and enriched. It is then prepared into liposomes containing pH-sensitive polyhistidine and modified with the dual ligand, enabling the drug to accurately identify autism brain regions. At the same time, the pH-sensitive properties promote the targeted release of the drug in the target area. Therefore, it achieves precise targeting and high efficacy, solving the problem in existing solutions for social and cognitive impairment in autism where the drug is widely distributed in non-target areas, resulting in insufficient effective concentration in the target area and difficulty in specifically improving pathological damage.
[0026] 2. This application employs a dual-ligand specific to the autism brain region. Through targeted enrichment in the affinity chromatography stage and targeted modification in the liposome formulation stage, a dual guarantee is formed, enabling the drug to accurately identify the corresponding receptors in key autism brain regions and achieve targeted enrichment. At the same time, the introduction of pH-sensitive polyhistidine into the liposomes allows the drug to respond to the pH characteristics of the inflammatory microenvironment in the target area, and release the effective ingredient in a targeted manner, avoiding ineffective distribution and waste of the drug in non-target areas, and improving the effective drug concentration in the target area.
[0027] 3. This application effectively maintains the structural integrity of liposomes during the lyophilization process by screening and optimizing the combination of lyophilization protectants and combining the parameters of gradient pre-freezing and vacuum lyophilization, thus avoiding drug leakage and loss of activity. At the same time, high-pressure homogenization ensures the uniformity of the liposome suspension, and the formulation performance remains stable after reconstitution, improving long-term storage stability, adapting to different storage and transportation environments, and reducing the risk of clinical use. Attached Figure Description
[0028] Figure 1 This is a flowchart illustrating the application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients, as provided in this application. Detailed Implementation
[0029] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.
[0030] Technical concept: Improving social and cognitive impairment in autism relies on drugs that can precisely intervene in the core pathological processes. However, in related technologies, drug delivery lacks specific recognition of key brain regions in autism, resulting in a large distribution of drugs in non-target areas and insufficient effective concentration in the target area, making it difficult to exert a targeted effect. At the same time, the preparation of natural active ingredients mostly relies on traditional methods, which have problems such as low synthesis efficiency, insufficient purity, and interference from by-products. Moreover, the formulations are mostly in liquid form, which has poor storage stability. These factors together restrict the actual application effect of the drugs.
[0031] To address the aforementioned issues, this application utilizes recombinant Saccharomyces cerevisiae strain modification and metabolic flux reprogramming fermentation to block precursor competition pathways, achieving efficient and high-quality synthesis of echinacoside. By employing dual modification with a dual-ligand specific to the autism brain region and a pH-sensitive liposome carrier, the drug is endowed with precise targeted recognition and targeted release capabilities. Furthermore, by combining gradient pre-freezing and vacuum freeze-drying processes, the long-term storage stability of the formulation is improved, resolving the issue of insufficient targeting and providing a reliable solution for improving social and cognitive impairments in autistic individuals.
[0032] Unless otherwise specified, all experimental methods used below are conventional methods. All materials, reagents, methods, and instruments used, unless otherwise specified, are conventional materials, reagents, methods, and instruments in this field, which can be obtained commercially or prepared according to literature methods by those skilled in the art.
[0033] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.
[0034] The following is a further description with reference to the embodiments: Example 1: Please refer to the appendix Figure 1 The application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients includes the following steps: A recombinant Saccharomyces cerevisiae strain was constructed, precursor competitive pathway genes were knocked out, and a precursor synthesis enhancement module containing a constitutive promoter and an echinacoside synthesis module containing an inducible promoter were introduced to obtain the recombinant strain; The recombinant strain was subjected to metabolic flux reprogramming fermentation, with pH controlled in stages at 6 and 5, and 0.4% methanol was added in a gradient to induce fermentation and obtain fermentation broth. The fermentation broth was centrifuged at 8000 r / min and pretreated with a 10 kDa ultrafiltration membrane to remove impurities, forming a pretreated solution. The pretreatment solution was purified by dual-target affinity chromatography, and echinacoside was obtained by gradient elution on a chromatography column coupled with a specific dual ligand for the autism brain region. Echinacea glycosides were prepared into liposomes containing pH-sensitive polyhistidine, which were then homogenized under high pressure at 800 bar and modified with dual ligands to obtain a suspension. By adding a lyophilization protectant to the suspension and performing gradient pre-freezing and vacuum lyophilization, a drug to improve social and cognitive impairment in autistic patients was obtained.
[0035] Constructing a recombinant Saccharomyces cerevisiae strain includes the following steps: The TYR1 and GSY1 genes of Saccharomyces cerevisiae were knocked out using CRISPR-Cas9 technology to obtain knockout yeast; The precursor synthesis enhancement module and the echinacea glycoside synthesis module were co-transferred into the knockout strain. Positive strains were obtained through uracil auxotrophic screening, forming a recombinant Saccharomyces cerevisiae strain. The precursor synthesis enhancement module contains the G6PDH gene and the PAL gene, and its expression is driven by the TEF1 constitutive promoter. The echinacea glycoside synthesis module contains the UGT76G1 gene and the CYP73A5 gene, and its expression is driven by the GAL1 inducible promoter.
[0036] Metabolic flux reprogramming fermentation of recombinant strains includes the following steps: The seed culture of the recombinant Saccharomyces cerevisiae strain was inoculated into the fermenter at an inoculation rate of 10% (v / v) and cultured at 31℃, 180r / min and an aeration rate of 1vvm. The pH was maintained at 6 for 0-24 hours during cultivation, and then adjusted to 5 after 24 hours. Methanol was added in three portions, 12 hours apart, with a single addition of 0.4% (v / v). The fermentation cycle was 72 hours. The concentration of echinacoside in the fermentation broth was 300 mg / L, and the content of tyrosol, a byproduct, was ≤8 mg / L.
[0037] The centrifugation time was 10 min, the pressure of ultrafiltration membrane pretreatment was 0.2 MPa, the removal rate of macromolecular impurities in the pretreatment solution was ≥95%, and the transmittance was ≥90%.
[0038] The process of obtaining echinacosides includes the following steps: The pretreatment solution was passed through a chromatography column containing a specific dual ligand for the autism brain region at a flow rate of 1 mL / min to bind echinacoside to the dual ligand. Impurities were eluted with Tris-HCl buffer containing 0.05 mol / L imidazole and pH 7.4, and then the bound echinacoside was eluted with Tris-HCl buffer containing 0.1 mol / L imidazole and pH 6.8. After elution, the echinacoside was purified by reversed-phase HPLC with methanol-water as the mobile phase (volume ratio 40:60) and a flow rate of 1 mL / min to obtain echinacoside with a purity ≥99.5%.
[0039] The brain region-specific dual ligands for autism are OXTR-Ab and 5-HTP, with a molar ratio of OXTR-Ab to the column carrier of chromatography of 1:50 and a molar ratio of OXTR-Ab to 5-HTP of 2:1.
[0040] To obtain the suspension, the following steps are included: PC, Chol and pH-sensitive polyhistidine were mixed in a mass ratio of 10:5:1.25 and then added to anhydrous ethanol at a mass ratio of 4 times. The mixture was stirred and dissolved in a water bath at 62.5°C for 20 minutes to prepare a liposome membrane material. Echinacoside was dissolved in physiological saline to prepare an aqueous solution of 10 mg / mL. Anhydrous ethanol was added to the liposome membrane material and dissolved by stirring in a water bath at 62.5°C to form an organic phase. Then, it was mixed with the aqueous phase solution at a volume ratio of 1:4 and stirred at 62.5°C for 30 min to form colostrum liposomes.
[0041] The dual ligands were modified after homogenization under high pressure at 800 bar, including the following steps: Colostrum liposomes were homogenized five times under high pressure at 800 bar to form a liposome suspension. Mix OXTR-Ab and 5-HTP at a molar ratio of 2:1 until homogeneous, add liposome suspension, and incubate at 37°C for 2 hours to allow the dual ligands to be covalently linked to the liposome surface, forming a suspension.
[0042] The freeze-drying protectant is a trehalose-mannitol complex, added at 10% of the suspension mass, with a trehalose to mannitol mass ratio of 3:1.
[0043] The gradient pre-freezing and vacuum freeze-drying process includes the following steps: The suspension with added freeze-drying protectant was cooled to -10°C at a rate of 5°C / min and held at that temperature for 1 hour. Then it was cooled to -40°C at a rate of 10°C / min and held at that temperature for 2 hours to form a pre-frozen product. The pre-frozen material was dried at a vacuum of 10 Pa and a temperature of -20 °C for 12 hours, and then dried at a temperature of 25 °C for 4 hours.
[0044] Example 2: This example differs from Example 1 above in that: The application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients includes the following steps: A recombinant Saccharomyces cerevisiae strain was constructed, precursor competitive pathway genes were knocked out, and a precursor synthesis enhancement module containing a constitutive promoter and an echinacoside synthesis module containing an inducible promoter were introduced to obtain the recombinant strain; The recombinant strain was subjected to metabolic flux reprogramming fermentation, with pH adjusted in stages to 6.2 and 5.2, and 0.6% methanol was added in a gradient to induce fermentation, and the fermentation broth was obtained. The fermentation broth was centrifuged at 9000 r / min and pretreated with a 12 kDa ultrafiltration membrane to remove impurities, forming a pretreated solution. The pretreatment solution was purified by dual-target affinity chromatography, and echinacoside was obtained by gradient elution on a chromatography column coupled with a specific dual ligand for the autism brain region. Echinacea glycosides were prepared into liposomes containing pH-sensitive polyhistidine, which were then homogenized under high pressure at 1000 bar and modified with dual ligands to obtain a suspension. By adding a lyophilization protectant to the suspension and performing gradient pre-freezing and vacuum lyophilization, a drug to improve social and cognitive impairment in autistic patients was obtained.
[0045] Example 3: This example differs from Example 1 above in that: The application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients includes the following steps: A recombinant Saccharomyces cerevisiae strain was constructed, precursor competitive pathway genes were knocked out, and a precursor synthesis enhancement module containing a constitutive promoter and an echinacoside synthesis module containing an inducible promoter were introduced to obtain the recombinant strain; The recombinant strain was subjected to metabolic flux reprogramming fermentation, with pH adjusted in stages to 5.8 and 4.8, and 0.2% methanol was added in a gradient to induce fermentation, and the fermentation broth was obtained. The fermentation broth was centrifuged at 7000 r / min and pretreated with an 8 kDa ultrafiltration membrane to remove impurities, forming a pretreated solution. The pretreatment solution was purified by dual-target affinity chromatography, and echinacoside was obtained by gradient elution on a chromatography column coupled with a specific dual ligand for the autism brain region. Echinacea glycosides were prepared into liposomes containing pH-sensitive polyhistidine, which were then homogenized under high pressure at 600 bar and modified with dual ligands to obtain a suspension. By adding a lyophilization protectant to the suspension and performing gradient pre-freezing and vacuum lyophilization, a drug to improve social and cognitive impairment in autistic patients was obtained.
[0046] Comparative Example 1: A method for preparing an adjunctive therapy to improve autism, comprising the following steps: Take the dried rhizome of Polygonum cuspidatum, pulverize it to 40 mesh, add 8 times the amount of 75% ethanol aqueous solution, and reflux at 80℃ twice, 2 hours each time, and combine the extracts; The extract was concentrated under reduced pressure to a relative density of 1.15 at 60°C. It was then suspended in 3 times the volume of distilled water and extracted 3 times with petroleum ether and ethyl acetate respectively. The ethyl acetate phase was collected, and the solvent was recovered under reduced pressure to obtain the crude extract. The crude extract was purified by silica gel column chromatography with petroleum ether-ethyl acetate as the eluent (volume ratio 3:1). The target elution peak was collected and dried under reduced pressure to obtain pure resveratrol. Weigh out microcrystalline cellulose, lactose, and magnesium stearate in a mass ratio of 1:3:1:0.05. Pure resveratrol was mixed evenly with microcrystalline cellulose and lactose, and a soft material was prepared by using 70% ethanol aqueous solution as a wetting agent. The material was granulated by 18-mesh sieve, dried at 60℃ for 2 hours, and then granulated by 16-mesh sieve. Add magnesium stearate and mix well. Fill the No. 0 hard capsule shells. Each capsule contains 20mg of resveratrol, which is the adjunctive treatment for autism.
[0047] Comparative Example 2: This comparative example differs from Example 1 above in that: The autism-specific dual ligand was replaced with the universal ligand bovine serum albumin (BSAL), with a BSAL molar ratio of 1:50 to the column carrier. After homogenization under high pressure (600-1000 bar), the dual ligands are modified, including the following steps: Colostrum liposomes were homogenized under high pressure of 600-1000 bar 4-6 times to form liposome suspensions; Add the universal ligand bovine serum albumin to the liposome suspension at a mass ratio of 1:100, and incubate at 37°C for 2 hours to form a suspension. The rest is the same as in Example 1.
[0048] Comparative Example 3: This comparative example differs from Example 1 above in that: The pH was adjusted in stages to 6.5 and 5.5, and the methanol replenishment was 0.7% (v / v) per batch. The rest is the same as in Example 1.
[0049] Performance testing: Brain region enrichment concentration: SPF-grade BTBR autism model mice were administered the drug via tail vein and sacrificed 2 hours later. The prefrontal cortex and amygdala tissues were collected, and the concentration of the active ingredient was detected by HPLC-MS. The enrichment factor was calculated with Comparative Example 1 as the baseline. Comparative Example 1 was administered by gavage, and the dose was converted to equivalent values. Social behavior detection: The model mice were placed in a three-box device, with an unfamiliar mouse on one side and an empty cage on the other side. The time the mice spent in the two boxes and the number of interactions were recorded within 10 minutes. The social preference index was calculated as the time spent in the unfamiliar mouse side / the total time spent in the box. Cognitive ability test: First, let the mice become familiar with two identical objects for 20 minutes. After 24 hours, replace one object with a new object. Record the exploration time of the mice for the old and new objects within 10 minutes, and calculate the recognition index: exploration time of the new object / total exploration time. Experimental period: The drug was administered continuously for 21 days, once a day, at a dose of 20 mg / kg. The blank control group was given an equal volume of physiological saline.
[0050] Table 1
[0051] As can be seen from Examples 1 to 3 and Comparative Example 1, and in conjunction with Table 1, this application achieves a synergistic effect throughout the entire process from strain construction and fermentation purification to formulation, making the acquisition of active ingredients more efficient and enabling precise drug delivery, thereby helping to improve social and cognitive related performance.
[0052] As can be seen from Examples 1 to 3 and Comparative Example 2, and in conjunction with Table 1, the autism brain region-specific dual ligand is an essential element for ensuring drug targeting. This dual ligand can guide the drug to be directionally enriched in key brain regions, ensuring that the targeting effect is fully exerted and providing support for the improvement of related manifestations.
[0053] As can be seen from Examples 1 to 3 and Comparative Example 3, and Table 1, reasonable control of the segmented pH and inducer addition rhythm during fermentation can ensure the efficient synthesis of active ingredients. The adaptability of parameters directly affects the overall effect of subsequent processes, and thus relates to the final improvement effect.
[0054] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment 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. The application of echinacoside in the preparation of drugs to improve social and cognitive impairment in autistic patients, characterized in that, Includes the following steps: A recombinant Saccharomyces cerevisiae strain was constructed, precursor competitive pathway genes were knocked out, and a precursor synthesis enhancement module containing a constitutive promoter and an echinacoside synthesis module containing an inducible promoter were introduced to obtain the recombinant strain; The recombinant strain was subjected to metabolic flux reprogramming fermentation, with the pH adjusted in stages to 5.8–6.2 and 4.8–5.2, and 0.2%–0.6% methanol was added in a gradient to induce fermentation and obtain the fermentation broth. The fermentation broth was centrifuged at 7000-9000 r / min and pretreated with an 8-12 kDa ultrafiltration membrane to remove impurities, forming a pretreated solution. The pretreatment solution was purified by dual-target affinity chromatography, and echinacoside was obtained by gradient elution on a chromatography column coupled with a specific dual ligand for the autism brain region. Echinacea glycosides were prepared into liposomes containing pH-sensitive polyhistidine, which were then homogenized under high pressure at 600-1000 bar and modified with dual ligands to obtain a suspension. By adding a lyophilization protectant to the suspension and performing gradient pre-freezing and vacuum lyophilization, a drug to improve social and cognitive impairment in autistic patients was obtained.
2. The application according to claim 1, characterized in that: The construction of the recombinant Saccharomyces cerevisiae strain includes the following steps: The TYR1 and GSY1 genes of Saccharomyces cerevisiae were knocked out using CRISPR-Cas9 technology to obtain knockout yeast; The precursor synthesis enhancement module and the echinacea glycoside synthesis module were co-transferred into the knockout strain. Positive strains were obtained through uracil auxotrophic screening to form a recombinant Saccharomyces cerevisiae strain. The precursor synthesis enhancement module contains the G6PDH gene and the PAL gene and is expressed using the TEF1 constitutive promoter. The echinacea glycoside synthesis module contains the UGT76G1 gene and the CYP73A5 gene and is expressed using the GAL1 inducible promoter.
3. The application according to claim 1, characterized in that: The metabolic flux reprogramming fermentation of the recombinant strain includes the following steps: The seed culture of the recombinant Saccharomyces cerevisiae strain was inoculated into the fermenter at an inoculation rate of 8%~12% (v / v) and cultured at 30~32℃, 160~200 r / min and an aeration rate of 0.8~1.2 vvm. During the first 0-24 hours of cultivation, the pH was maintained at 5.8-6.
2. After 24 hours, the pH was adjusted to 4.8-5.
2. Methanol was added in three separate additions at 12-hour intervals, with each addition being 0.2%-0.6% (v / v). The fermentation cycle was 68-76 hours. The concentration of echinacoside in the fermentation broth was 200-400 mg / L, and the content of tyrosol, a byproduct, was ≤8 mg / L.
4. The application according to claim 1, characterized in that: The centrifugation time is 8-12 min, the pressure of the ultrafiltration membrane pretreatment is 0.1-0.3 MPa, the removal rate of macromolecular impurities in the pretreatment solution is ≥95%, and the transmittance is ≥90%.
5. The application according to claim 1, characterized in that: The process of obtaining echinacea glycosides includes the following steps: The pretreatment solution was passed through a chromatography column containing a specific dual ligand for the autism brain region at a flow rate of 0.8–1.2 mL / min to bind echinacoside to the dual ligand. Impurities were eluted with Tris-HCl buffer containing 0.04–0.06 mol / L imidazole at pH 7.2–7.6, and then the bound echinacoside was eluted with Tris-HCl buffer containing 0.08–0.12 mol / L imidazole at pH 6.6–7.
0. After elution, the echinacoside was purified by reversed-phase HPLC with methanol-water as the mobile phase (volume ratio 35:65~45:55) and a flow rate of 0.8~1.2 mL / min to obtain echinacoside with a purity ≥99.5%.
6. The application according to claim 5, characterized in that: The autism brain region-specific dual ligands are OXTR-Ab and 5-HTP, with the molar ratio of OXTR-Ab to the chromatography column carrier being 1:40 to 1:60, and the molar ratio of OXTR-Ab to 5-HTP being 1.5:1 to 2.5:
1.
7. The application according to claim 1, characterized in that: The process of obtaining the suspension includes the following steps: PC, Chol and pH-sensitive polyhistidine in a mass ratio of 8~12:4~6:1~1.5 were added to anhydrous ethanol at 3~5 times their total mass and stirred in a water bath at 60~65℃ for 15~25 min to prepare liposome membrane material. Echinacoside was dissolved in physiological saline to prepare an aqueous solution of 8-12 mg / mL. Anhydrous ethanol is added to the liposome membrane material and dissolved by stirring in a water bath at 60-65°C to form an organic phase. Then, it is mixed with the aqueous phase solution at a volume ratio of 1:3 to 1:5 and stirred at 60-65°C for 25-35 minutes to form colostrum liposomes.
8. The application according to claim 1, characterized in that: The modification of the dual ligands after homogenization under high pressure (600-1000 bar) includes the following steps: Colostrum liposomes were homogenized under high pressure of 600-1000 bar 4-6 times to form liposome suspensions; Mix OXTR-Ab and 5-HTP at a molar ratio of 1.5:1 to 2.5:1 until homogeneous, add liposome suspension, and incubate at 35 to 39°C for 1.5 to 2.5 hours to allow the dual ligands to be covalently linked to the liposome surface, forming a suspension.
9. The application according to claim 1, characterized in that: The freeze-drying protectant is a trehalose-mannitol complex, added at 8% to 12% of the suspension mass, with a trehalose to mannitol mass ratio of 2.5:1 to 3.5:
1.
10. The application according to claim 1, characterized in that: The gradient pre-freezing and vacuum freeze-drying process includes the following steps: The suspension with added freeze-drying protectant was cooled to -8 to -12°C at a rate of 4 to 6°C / min and held at that temperature for 0.8 to 1.2 hours. Then it was cooled to -38 to -42°C at a rate of 8 to 12°C / min and held at that temperature for 1.5 to 2.5 hours to form a pre-frozen product. The pre-frozen material was dried under a vacuum of 8-12 Pa and a temperature of -22 to -18 °C for 10-14 hours, and then dried at a temperature of 23-27 °C for 3-5 hours.