Composition containing pyruvate and preparation method and application thereof

Abstract

The invention relates to the field of biological medicines, in particular to a composition containing pyruvate as well as a preparation method and application of the composition. The composition containing pyruvate is prepared from the following raw materials in percentage by mass: 0.05 to 0.1 percent of pyruvate, 0.02 to 0.04 percent of bergenin derivative, 5 to 10 percent of water for injection and the balance of propellant, the structural formula of the bergenin derivative is as follows: # imgabs0 #. The composition containing pyruvate can reduce the abundance of pathogenic bacteria and increase the abundance of probiotics, so that the effect of treating lung diseases is achieved by adjusting the respiratory tract micro-ecology.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a composition containing pyruvate, a preparation method thereof, and an application thereof. Background Art

[0002] The respiratory tract microecology refers to the complex ecosystem formed by the microbial community in the respiratory tract and its interaction with the host, which has dynamic characteristics and is constantly undergoing component migration, elimination, and reproduction. In recent years, research has shown that there is a complex interaction between the respiratory tract microecology and various respiratory diseases. The occurrence and development of diseases such as chronic obstructive pulmonary disease, lung cancer, and asthma are closely related to the imbalance of respiratory tract microorganisms. For example, in patients with chronic obstructive pulmonary disease, the composition of lung microorganisms is significantly different from that of healthy people, and the commonly increased microorganisms include Haemophilus parainfluenzae and Pseudomonas aeruginosa, and the increase of these microorganisms is related to the severity of the disease. In addition, the lung microbial diversity of patients with pulmonary fibrosis is usually low, and the proportion of specific microorganisms such as Streptococcus is increased, and this change is closely related to the pathological changes of lung tissue and may promote the fibrosis process. In summary, the research on the respiratory tract microecology provides a new perspective for us to understand and treat various respiratory diseases.

[0003] Research has found that sodium pyruvate can reduce pulmonary inflammation and congestion in patients with chronic non-obstructive pulmonary disease, pulmonary fibrosis, cystic fibrosis, asthma, sinusitis, and influenza. Inhalation of sodium pyruvate can also relieve symptoms related to CoVID-19 patients in pulmonary fibrosis. Patent CN118903009A uses a sodium pyruvate aerosol to regulate the respiratory tract microecology and obtains good results. However, in order to overcome the problems of unstable sodium pyruvate and poor drug efficacy, sodium pyruvate has been modified in 6 steps, resulting in complex preparation processes and high production costs. To solve the above problems, the present invention provides a composition containing pyruvate for regulating the respiratory tract microecology, which has a simple preparation process, good drug efficacy, and can greatly reduce costs. Summary of the Invention

[0004] To overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a composition containing pyruvate, which can significantly reduce the abundance of pathogenic bacteria and increase the abundance of probiotics.

[0005] Another purpose of the present invention is to provide a preparation method of a composition containing pyruvate, and the preparation steps are concise.

[0006] Another purpose of the present invention is to provide an application of a composition containing pyruvate, and the application prospect is broad.

[0007] One of the purposes of the present invention is achieved by adopting the following technical solutions: A composition containing pyruvate, by mass percentage, comprises the following raw materials: pyruvate 0.05 - 0.1%, bergenin derivative 0.02 - 0.04%, water for injection 5 - 10%, and the balance is propellant; the structural formula of the bergenin derivative is as follows: 。

[0008] Further, the preparation method of the bergenin derivative is as follows: (1) Add bergenin to DMF, then add potassium carbonate and 4-(chloromethyl)benzaldehyde, stir at room temperature, and then add hydrochloric acid for quenching reaction, and obtain intermediate 1 through purification; (2) Add intermediate 1 from step (1) to methanol, and then successively add baicalein and dimethylamine solution for reaction. After the reaction is completed, obtain intermediate 2 through purification; (3) Add intermediate 2 from step (2) and bromopropane to ethanol for reflux reaction, and obtain the bergenin derivative after purification.

[0009] Further, in step (1), the molar ratio of bergenin, 4-(chloromethyl)benzaldehyde, and potassium carbonate is 2:(4 - 5):(6 - 7); the concentration of the hydrochloric acid is 6 - 7 mol / L.

[0010] Further, the stirring time in step (1) is 12 - 16 h.

[0011] Further, in step (2), the molar ratio of intermediate 1, baicalein, and dimethylamine is 2:(4 - 5):(6 - 8); the concentration of the dimethylamine solution is 40 - 50%.

[0012] Further, the reaction temperature in step (2) is 30 - 70 °C.

[0013] Further, in step (3), the molar ratio of intermediate 2 from step (2) and bromopropane is 1:(2 - 3); the reflux reaction time is 50 - 55 h.

[0014] Further, the pyruvate is sodium pyruvate; the propellant is tetrafluoroethane or heptachloropropane.

[0015] The second object of the present invention is achieved by the following technical solution: The preparation method of the above composition containing pyruvate comprises the following steps: According to the mass percentage, compound pyruvate, bergenin derivative, and water for injection, fill, and press in the propellant to obtain.

[0016] The third object of the present invention is achieved by the following technical solution: Use of the above composition containing pyruvate in the preparation of a medicament for regulating the respiratory tract microecology.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The composition containing pyruvate obtained in the present invention contains components such as bergenin derivatives. Among them, the bergenin derivative is formed by reacting bergenin with 4-(chloromethyl)benzaldehyde to generate an intermediate 1 containing an aldehyde group, and then using intermediate 1 to carry out a Mannich reaction with baicalein and dimethylamine. In the bergenin derivative of the present invention, by introducing a quaternary ammonium cation, the electrostatic interaction with the pyruvate ion is enhanced to improve the stability of pyruvate; on the other hand, by introducing baicalein and bergenin, the abundance of pathogenic bacteria is reduced and the abundance of probiotics is increased, so as to achieve the effect of treating lung diseases by regulating the respiratory tract microecology.

[0018] 2. The preparation method of the composition containing pyruvate provided by the present invention is simple, efficient and low-cost. Detailed implementation manners

[0019] The following combines specific implementation manners to further describe the present invention. It should be noted that, on the premise of no conflict, the following described embodiments or technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0020] 1. Embodiment Embodiment 1 This embodiment provides a composition containing pyruvate, which is composed of the following raw materials by mass percentage: sodium pyruvate 0.08%, bergenin derivative 0.02%, water for injection 8%, and the balance is tetrafluoroethane; the structural formula of the bergenin derivative is as follows: .

[0021] The preparation method of the above bergenin derivative is as follows: (1) According to the dosage ratio of bergenin, 4-(chloromethyl)benzaldehyde, potassium carbonate, and DMF of 2 mmol: 4.5 mmol: 6 mmol: 25 mL; add bergenin to DMF, then add potassium carbonate and 4-(chloromethyl)benzaldehyde, stir at room temperature for 14 h, then add 6 mol / L hydrochloric acid for quenching. The reaction solution is extracted with ethyl acetate, and the ethyl acetate phase is washed with distilled water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and then separated and purified by column chromatography to obtain intermediate 1; The NMR results of intermediate 1 are as follows: Intermediate 1's 1 HNMR: (C 28 H 24 O 11 , 400 MHz, DMSO- d6 ) δ: 3.50 - 3.61 (m, 4H), 3.73 (s, 3H), 3.95 (s, 1H), 4.27 - 4.30 (m, 1H), 4.52 (s, 2H), 5.13 - 5.16 (m, 1H), 5.46 - 5.48 (d, 1H), 7.31 (s, 1H), 7.35 - 7.37 (d, 4H), 8.01 - 8.03 (d, 4H), 9.91 (s, 2H), MS(ESI) m / z = 537.13 [M + H] + , found 537.14; the above results confirm that the obtained product is the target product.

[0022] (2) According to the dosage ratio of intermediate 1, baicalein, dimethylamine, and methanol of 2 mmol: 4.5 mmol: 7 mmol: 60 mL, add the intermediate 1 from step (1) to methanol, then successively add baicalein and a 40% dimethylamine solution, react at 50 °C, monitor the reaction progress by thin-layer chromatography. After the reaction is completed, vacuum filter to obtain a solid product, wash it with a small amount of methanol, and then dry it in a vacuum oven to obtain intermediate 2.

[0023] The NMR results of intermediate 2 are as follows: 1 HNMR (C 62 H 54 N 2 O 19 , 400 MHz, DMSO- d6δ: 2.26 (s, 12H), 3.50 - 3.61 (m, 4H), 3.71 (s, 3H), 3.94 (s, 1H), 4.28 - 4.30 (m, 1H), 4.51 (s, 2H), 5.13 - 5.16 (m, 3H), 5.46 - 5.48 (d, 1H), 6.71 (s, 2H), 7.16 - 7.18 (d, 4H), 7.32 (s, 1H), 7.35 - 7.37 (d, 4H), 7.45 - 7.49 (m, 6H), 7.76 - 7.78 (m, 4H), 8.73 (s, 2H), 9.58 (s, 2H), MS(ESI) m / z = 1131.33 [M + H] + , found 1131.33; the above results confirm that the obtained product is the target product.

[0024] (3) According to the dosage ratio of intermediate 2, bromopropane, and ethanol in step (2) of 1 mmol: 2.5 mmol: 40 mL, add intermediate 2 and bromopropane in step (2) to ethanol and reflux for 52 h, filter to obtain a solid product, and recrystallize with ethanol to obtain the bergenin derivative.

[0025] The NMR results of the bergenin derivative are as follows: 1 HNMR (C 68 H 68 N 2 O 19 Br 2 , 400 MHz, DMSO - d6 δ: 0.93 - 0.95 (t, 6H), 1.75 - 1.79 (m, 4H), 3.22 (t, 4H), 3.30 (s, 12H), 3.50 - 3.61 (m, 4H), 3.71 (s, 3H), 3.94 (s, 1H), 4.28 - 4.30 (m, 1H), 4.51 (s, 2H), 5.15 (t, 1H), 5.46 - 5.48 (d, 1H), 6.14 (s, 2H), 6.71 (s, 2H), 7.16 - 7.18 (d, 4H), 7.31 - 7.33 (m, 4H), 7.45 - 7.49 (m, 6H), 7.76 - 7.78 (m, 4H), 8.73 (s, 2H), 9.58 (s, 2H); the above results confirm that the obtained product is the target product.

[0026] This example also provides a preparation method of the above composition containing pyruvate, including the following steps: According to the mass percentage, compound sodium pyruvate, bergenin derivative, and water for injection, fill, and press in tetrafluoroethane to obtain it.

[0027] Example 2 This example provides a composition containing pyruvate, which is composed of the following raw materials by mass percentage: sodium pyruvate 0.05%, bergenin derivative 0.03%, water for injection 5%, and the balance is heptachloropropane; the structural formula of the bergenin derivative is the same as that in Example 1.

[0028] The preparation method of the above bergenin derivative is as follows: (1) According to the dosage ratio of bergenin, 4-(chloromethyl)benzaldehyde, potassium carbonate, and DMF of 2 mmol: 4 mmol: 6 mmol: 25 mL; add bergenin to DMF, then add potassium carbonate and 4-(chloromethyl)benzaldehyde, stir at room temperature for 12 h, then add 6 mol / L hydrochloric acid for quenching, the reaction solution is extracted with ethyl acetate, and the ethyl acetate phase is washed with distilled water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and then separated and purified by column chromatography to obtain Intermediate 1; the 1 HNMR and MS(ESI) m / z of Intermediate 1 are consistent with those in Example 1.

[0029] (2) According to the dosage ratio of Intermediate 1, baicalein, dimethylamine, and methanol of 2 mmol: 4 mmol: 6 mmol: 60 mL, add Intermediate 1 in step (1) to methanol, then successively add baicalein and a 40% dimethylamine solution, react at 30 °C, monitor the reaction progress by thin layer chromatography, after the reaction is completed, vacuum filter to obtain a solid product, wash with a small amount of methanol, and then dry in a vacuum oven to obtain Intermediate 2. The 1 HNMR and MS(ESI) m / z of Intermediate 2 are consistent with those in Example 1.

[0030] (3) According to the dosage ratio of Intermediate 2 in step (2), bromopropane, and ethanol of 1 mmol: 2 mmol: 30 mL, add Intermediate 2 and bromopropane in step (2) to ethanol and reflux for 50 h, filter to obtain a solid product, and recrystallize with ethanol to obtain the bergenin derivative. The 1 HNMR of the bergenin derivative is consistent with that in Example 1.

[0031] This example also provides a preparation method of the above composition containing pyruvate, which includes the following steps: According to the mass percentage, compound sodium pyruvate, bergenin derivative and water for injection, fill, and press in heptachloropropane to obtain.

[0032] Example 3 This embodiment provides a composition containing pyruvate, which is composed of the following raw materials by mass percentage: sodium pyruvate 0.1%, bergenin derivative 0.04%, water for injection 10%, and the balance is tetrafluoroethane; the structural formula of the bergenin derivative is the same as that in Example 1.

[0033] The preparation method of the above bergenin derivative is as follows: (1) According to the dosage ratio of bergenin, 4-(chloromethyl)benzaldehyde, potassium carbonate, and DMF of 2 mmol: 5 mmol: 6 mmol: 25 mL; add bergenin to DMF, then add potassium carbonate and 4-(chloromethyl)benzaldehyde, stir at room temperature for 16 h, then add 6 mol / L hydrochloric acid for quenching, the reaction solution is extracted with ethyl acetate, the ethyl acetate phase is washed with distilled water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and then separated and purified by column chromatography to obtain Intermediate 1; the 1 HNMR and MS(ESI) m / z of Intermediate 1 are consistent with those in Example 1.

[0034] (2) According to the dosage ratio of Intermediate 1, baicalein, dimethylamine, and methanol of 2 mmol: 5 mmol: 8 mmol: 60 mL, add Intermediate 1 from step (1) to methanol, then successively add baicalein and a 40% dimethylamine solution, react at 70 °C, monitor the reaction progress by thin-layer chromatography, after the reaction is completed, filter under vacuum to obtain a solid product, wash with a small amount of methanol, and then dry in a vacuum oven to obtain Intermediate 2. The 1 HNMR and MS(ESI) m / z of Intermediate 2 are consistent with those in Example 1.

[0035] (3) According to the dosage ratio of Intermediate 2 from step (2), bromopropane, and ethanol of 1 mmol: 3 mmol: 50 mL, add Intermediate 2 and bromopropane from step (2) to ethanol and reflux for 55 h, filter to obtain a solid product, and recrystallize with ethanol to obtain the bergenin derivative. The 1 HNMR of the bergenin derivative is consistent with that in Example 1.

[0036] This embodiment also provides a preparation method of the above composition containing pyruvate, including the following steps: According to the mass percentage, compound sodium pyruvate, bergenin derivative, and water for injection, fill, and press in tetrafluoroethane to obtain the product.

[0037] 2. Comparative Examples Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that bergenin is used to replace the bergenin derivative, and the rest is the same as in Example 1.

[0038] Comparative Example 2 The difference between Comparative Example 2 and Example 1 lies in that the bergenin derivative is omitted, and at the same time, the dosage of sodium pyruvate is increased to 0.1%, and the rest is the same as in Example 1.

[0039] The effects of the drugs obtained in the present invention are described below.

[0040] I. Animal experiments Construction of a rat model of pulmonary fibrosis and administration method Experimental animals: 70 SD rats, each rat weighing 210 - 220 g. All rats were randomly divided into 7 groups, with the male - female ratio of 1:1 in each group. After 5 days of adaptive feeding, one of the groups was randomly selected as the blank control group.

[0041] Model construction: Each group of rats was anesthetized by intraperitoneal injection of 4% chloral hydrate, and the injection volume of chloral hydrate was 10 mL / kg. Then, the anesthetized rats in each group were fixed in a supine position, the hair on the neck was removed, and a longitudinal incision was made to expose the trachea. Except for the blank control group, rats in other groups were inserted into the trachea 1 cm towards the heart end through the gap between two tracheal cartilage rings using a syringe. After no resistance was aspirated back, bleomycin (dose: 5 mg / kg) was injected into the trachea, and the blank control group was injected with an equal volume of normal saline. After injection, each group of rats was rotated upright for 3 minutes to evenly distribute the drug in both lungs, and each layer of tissue was sutured.

[0042] Administration method: 24 hours after model establishment, rats in the blank group and the model group were intragastrically administered an equal amount of normal saline, and rats in Example 1 - 3 and Comparative Example 1 - 2 groups were given each preparation at 0.6 mg / kg (i.e., the dosage of sodium pyruvate was 0.6 mg / kg) by atomization, and the administration was continued for 28 days.

[0043] Detection indexes and detection methods: (1) Body weight: The body weight of rats in each group was measured on the day of administration and recorded as the initial weight. The body weight of rats in each group was measured again every 7 days. The experimental result of each group was the average value of the experimental results of all rats in each group. The results are shown in Table 1.

[0044] (2) Lung coefficient: On the 14th day and the 28th day, 5 rats in each group were randomly sacrificed, and then the lung tissues of each rat were taken for weighing to calculate the lung coefficient. Lung coefficient = wet lung weight (mg) / body weight (mg) × 100%. The experimental result was the average value of the experimental results of all rats in each group. The results are shown in Table 2.

[0045] (3) Hydroxyproline content in lung tissue: After 28 days of administration, the remaining 5 rats in each group were sacrificed. After calculating the lung coefficient, the hydroxyproline content in the lung tissue was detected using a hydroxyproline content detection kit. The experimental result was the average value of the experimental results of all rats in each group. The results are shown in Table 2.

[0046] Table 1 Table 2 As can be seen from Table 1, compared with the blank control group, the body weight of the rats in the model group decreased significantly on the 21st day. Compared with the model group, the body weights of the rats in Examples 1-3 and Comparative Examples 1-2 increased, and the increase in the body weights of the rats in Examples 1-3 was more significant. In Comparative Example 1, bergenin was replaced with bergenin derivative, and in Comparative Example 2, the bergenin derivative was omitted, but the dosage of sodium pyruvate was increased. The above results indicate that the composition containing pyruvate obtained in the present invention can effectively alleviate the impact of pulmonary fibrosis on rats.

[0047] As can be seen from Table 2, compared with the blank control group, the lung coefficient and the content of hydroxyproline in the lung tissue of the rats in the model group increased significantly. After administration, compared with the model group, the lung coefficient and the content of hydroxyproline in the lung tissue of the rats in Examples 1-3 and Comparative Examples 1-2 decreased, and the decrease in the lung coefficient and the content of hydroxyproline in the lung tissue of the rats in Examples 1-3 was particularly obvious. The above results indicate that the composition containing pyruvate obtained in the present invention can effectively improve pulmonary fibrosis in rats. That is, the composition containing pyruvate obtained in the present invention can effectively alleviate the development degree of pulmonary fibrosis in model rats.

[0048] II. Clinical Experiment Experimental Process and Administration Method Experimental Subjects: According to the medical history and clinical relevant treatments of the patients, a total of 25 patients with acute exacerbation of COPD were included in the present invention.

[0049] Administration Method: After the patients were discharged from the hospital, the composition containing pyruvate obtained in Example 1 was used once a day, and the patients were followed up for 6 months.

[0050] Experimental Process and Experimental Results: The nucleic acid was extracted using the TGuide S96 bead method lavage fluid genomic DNA extraction kit, and the extract was detected using the 16s rRNA detection technology. Primer Design: For bacteria, the universal bacterial primer 16s was used to amplify the V3+V4 region of the 16s rDNA gene of the sample; the amplification region for the fungi in the sample was the ITS1 region, and the primer design is shown in Table 3: Table 3 The types and abundances of the flora of the present invention were confirmed by comparing with the known sequences in the database, and the results are shown in Tables 4-5.

[0051] Table 4 Bacterial Community Abundances at the Phylum Level Before and After Medication Table 5 Bacterial Community Abundances at the Genus Level Before and After Medication As can be seen from Table 4, after using the composition containing pyruvate obtained by the present invention, Cynaobacteria Cyanobacteria, Actinobacteria Actinobacteria, Firmicutes Firmicutes have increased abundances, Proteobacteria Proteobacteria, Bacteroidetes Bacteroidetes, Fusobacteria Fusobacteria have decreased abundances.

[0052] As can be seen from Table 5, after using the composition containing pyruvate obtained by the present invention, Haemophilus Haemophilus has a significantly decreased abundance, while Streptococcus Streptococcus, Lactobacillus Lactobacillus have increased abundances.

[0053] In summary, after using the composition containing pyruvate obtained by the present invention, the content of Firmicutes has increased significantly, including the significantly increased contents of Streptococcus and Lactobacillus at the genus level; the content of Proteobacteria has decreased significantly, including the significantly decreased content of Haemophilus at the genus level. In summary, the composition containing pyruvate obtained by the present invention can reduce the abundance of pathogenic bacteria and increase the abundance of probiotic bacteria.

[0054] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention belong to the scope of protection required by the present invention.

Claims

1. A composition comprising pyruvate, characterized in that The raw materials are as follows: 0.05-0.1% pyruvate, 0.02-0.04% bergenia lactone derivative, 5-10% water for injection, and the balance is propellant; the structural formula of the bergenia lactone derivative is as follows: 。 2. The composition comprising pyruvate according to claim 1, characterized in that The preparation method of the bergenia lactone derivative is as follows: (1) Add bergenia lactone to DMF, then add potassium carbonate and 4-(chloromethyl)benzaldehyde, stir at room temperature, then add hydrochloric acid to quench the reaction, and purify to obtain intermediate 1; (2) adding the intermediate 1 of step (1) into methanol, and then sequentially adding baicalein and dimethylamine solution to react, and after the reaction is completed, purifying to obtain the intermediate 2; (3) The intermediate 2 of step (2) and bromopropane are added to ethanol for reflux reaction, and the bergenia lactone derivative is obtained after purification.

3. The composition comprising pyruvate according to claim 2, characterized in that In step (1), the molar ratio of bergenia lactone, 4-(chloromethyl)benzaldehyde and potassium carbonate is 2:(4-5):(6-7); the concentration of hydrochloric acid is 6-7 mol / L.

4. The composition comprising pyruvate according to claim 2, characterized in that The stirring time in step (1) is 12-16 hours.

5. The composition comprising pyruvate according to claim 2, characterized in that In step (2), the molar ratio of the intermediate 1, baicalein and dimethylamine is 2:(4-5):(6-8); the concentration of the dimethylamine solution is 40-50%.

6. The composition comprising pyruvate according to claim 2, characterized in that The reaction temperature in step (2) is 30-70°C.

7. The composition comprising pyruvate according to claim 2, characterized in that In step (3), the molar ratio of the intermediate 2 in step (2) to bromopropane is 1:(2-3); and the reflux reaction time is 50-55h.

8. The composition comprising pyruvate according to claim 1, characterized in that The pyruvate is sodium pyruvate; the propellant is tetrafluoroethane or heptachloropropane.

9. A method for preparing a composition comprising pyruvate according to any one of claims 1 to 8, characterized in that: The following steps are involved: The pyruvate, the bergenia lactone derivative and the water for injection are compounded according to the mass percentage, filled and pressed into the propellant to obtain the product.

10. Use of the composition comprising pyruvate according to any one of claims 1 to 8 in the preparation of a medicament for regulating respiratory microecology.

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