A composition comprising pyruvate, its preparation method and application

Abstract

This invention relates to the field of biomedicine, and more particularly to a composition comprising pyruvate, its preparation method, and its application. The composition comprising pyruvate, by mass percentage, includes the following raw materials: 0.05-0.1% pyruvate, 0.02-0.04% Bergenin derivative, 5-10% water for injection, and the balance being a propellant; the structural formula of the Bergenin derivative is shown below. The above-mentioned composition comprising pyruvate can reduce the abundance of pathogenic bacteria and increase the abundance of probiotics, thereby achieving the effect of treating lung diseases by regulating the respiratory microecology.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a composition comprising pyruvate, its preparation method, and its application. Background Technology

[0002] The respiratory tract microbiota refers to the complex ecosystem formed by the microbial community within the respiratory tract and its interaction with the host. It is dynamic, with components constantly migrating, being eliminated, and multiplying. Recent studies have shown a complex interaction between the respiratory tract microbiota and various respiratory diseases. The development of diseases such as chronic obstructive pulmonary disease (COPD), lung cancer, and asthma are closely related to respiratory microbial imbalances. For example, the composition of lung microbiota in COPD patients differs significantly from that in healthy individuals, with commonly increased microorganisms including *Streptococcus harveyi* and *Pseudomonas*. This increase is correlated with disease severity. Furthermore, patients with pulmonary fibrosis typically have lower lung microbial diversity, with an increased proportion of specific microorganisms such as *Streptococcus*. This change is closely related to pathological changes in lung tissue and may promote the fibrotic process. In summary, research on the respiratory tract microbiota provides new perspectives for understanding and treating various respiratory diseases.

[0003] Studies have found that sodium pyruvate can reduce lung inflammation and congestion in patients with chronic non-obstructive lung disease, pulmonary fibrosis, cystic fibrosis, asthma, sinusitis, and influenza. Inhaled sodium pyruvate can also alleviate symptoms associated with COVID-19 in patients with pulmonary fibrosis. Patent CN118903009A achieved good results using sodium pyruvate aerosol to regulate the respiratory microecology; however, to overcome the instability and poor efficacy of sodium pyruvate, a six-step modification process was performed, resulting in a complex preparation process and high production costs. To address these issues, this invention provides a composition containing pyruvate salt for regulating the respiratory microecology. This composition is simple to prepare, has good efficacy, and can significantly reduce costs. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a composition containing pyruvate that can significantly reduce the abundance of pathogenic bacteria and increase the abundance of probiotics.

[0005] A second objective of this invention is to provide a method for preparing a composition containing pyruvate, which has a simple preparation process.

[0006] The third objective of this invention is to provide an application of a composition containing pyruvate, which has broad application prospects.

[0007] One of the objectives of this invention is achieved through the following technical solution:

[0008] A composition comprising pyruvate, comprising, by weight percentage: 0.05-0.1% pyruvate, 0.02-0.04% Bergenin derivative, 5-10% water for injection, and the balance being a propellant; the structural formula of the Bergenin derivative is shown below:

[0009] .

[0010] Furthermore, the preparation method of the Bergenia lactone derivative is as follows:

[0011]

[0012] (1) Bergenin was added to DMF, then potassium carbonate and 4-chlorobenzaldehyde were added, stirred at room temperature, and then hydrochloric acid was added to quench the reaction. After purification, intermediate 1 was obtained.

[0013] (2) Add intermediate 1 from step (1) to methanol, and then add baicalein and dimethylamine solution in sequence to react. After the reaction is completed, purify to obtain intermediate 2.

[0014] (3) The intermediate 2 and bromopropane from step (2) were added to ethanol and refluxed to obtain Bergenia lactone derivative after purification.

[0015] Further, in step (1), the molar ratio of Bergenin, 4-chlorobenzaldehyde, and potassium carbonate is 2:(4-5):(6-7); and the concentration of hydrochloric acid is 6-7 mol / L.

[0016] Furthermore, the stirring time in step (1) is 12-16 hours.

[0017] Further, in step (2), the molar ratio of intermediate 1, baicalein, and dimethylamine is 2:(4-5):(6-8); and the concentration of the dimethylamine solution is 40-50%.

[0018] Furthermore, the reaction temperature in step (2) is 30-70℃.

[0019] Further, in step (3), the molar ratio of intermediate 2 and bromopropane in step (2) is 1:(2-3); the reflux reaction time is 50-55h.

[0020] Furthermore, the pyruvate is sodium pyruvate; the propellant is tetrafluoroethane or heptachloropropane.

[0021] The second objective of this invention is achieved by the following technical solution:

[0022] The method for preparing the above-mentioned composition containing pyruvate includes the following steps:

[0023] The pyruvate, Bergenin lactone derivative and water for injection are compounded according to the mass percentage, filled and injected with propellant to obtain the final product.

[0024] The third objective of this invention is achieved by the following technical solution:

[0025] The above-mentioned composition containing pyruvate is used in the preparation of a medicament for regulating the respiratory microecology.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. The composition containing pyruvate obtained by the present invention includes components such as Bergenin lactone derivatives. The Bergenin lactone derivative is obtained by reacting Bergenin lactone with 4-chlorobenzaldehyde to generate an intermediate 1 containing an aldehyde group, followed by a Mannich reaction between intermediate 1 and baicalein and dimethylamine. The Bergenin lactone derivative of the present invention improves the stability of pyruvate by introducing quaternary ammonium cations to enhance their electrostatic interaction with pyruvate ions; on the other hand, it reduces the abundance of pathogenic bacteria and increases the abundance of probiotics by introducing baicalein and Bergenin lactone, thereby achieving the effect of treating lung diseases by regulating the respiratory microecology.

[0028] 2. The preparation method of the composition containing pyruvate provided by the present invention is simple, efficient and low cost. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments shall be performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0030] 1. Example

[0031] Example 1

[0032] This embodiment provides a composition comprising pyruvate, which, by mass percentage, consists of the following raw materials: 0.08% sodium pyruvate, 0.02% Bergenin derivative, 8% water for injection, and the balance being tetrafluoroethane; the structural formula of the Bergenin derivative is shown below:

[0033] .

[0034] The preparation method of the above-mentioned Bergenia lactone derivatives is as follows:

[0035]

[0036] (1) Bergenin, 4-chlorobenzaldehyde, potassium carbonate and DMF were added in a ratio of 2 mmol: 4.5 mmol: 6 mmol: 25 mL. Bergenin was added to DMF, then potassium carbonate and 4-chlorobenzaldehyde were added. The mixture was stirred at room temperature for 14 h, then quenched with 6 mol / L hydrochloric acid. The reaction solution was extracted with ethyl acetate, and the ethyl acetate phase was washed with distilled water and saturated brine, dried with anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain intermediate 1.

[0037] The NMR results for intermediate 1 are as follows:

[0038] Intermediate 1 1 HNMR: (C 28 H 24 O 11 400MHz, 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] + 537.14 was found; the above results confirm that the obtained product is the target product.

[0039] (2) According to the ratio of intermediate 1, baicalin, dimethylamine and methanol, 2 mmol: 4.5 mmol: 7 mmol: 60 mL, intermediate 1 from step (1) was added to methanol, and then baicalin and a 40% dimethylamine solution were added in sequence. The reaction was carried out at 50 °C. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the solid product was obtained by vacuum filtration, washed with a small amount of methanol, and then dried in a vacuum oven to obtain intermediate 2.

[0040] The NMR results for intermediate 2 are as follows:

[0041] 1 HNMR (C 62 H 54 N2O 19 400MHz, DMSO- d6) δ: 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] + The result found 1131.33; the above results confirm that the obtained product is the target product.

[0042] (3) According to the ratio of intermediate 2, bromopropane and ethanol in step (2) of 1 mmol: 2.5 mmol: 40 mL, intermediate 2 and bromopropane in step (2) were added to ethanol and refluxed for 52 h. The solid product was obtained by filtration and recrystallization with ethanol to obtain Bergenia lactone derivative.

[0043] The NMR results of Bergenia lactone derivatives are as follows:

[0044] 1 HNMR (C 68 H 68 N2O 19 Br2, 400MHz, 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),5.4 6-5.48 (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.

[0045] This embodiment also provides a method for preparing the above-mentioned composition containing pyruvate, comprising the following steps:

[0046] Sodium pyruvate, Bergenin lactone derivative, and water for injection are compounded according to the mass percentage, filled into containers, and then injected with tetrafluoroethane to obtain the final product.

[0047] Example 2

[0048] This embodiment provides a composition containing pyruvate, which, by mass percentage, consists of the following raw materials: 0.05% sodium pyruvate, 0.03% Bergenin derivative, 5% water for injection, and the balance being heptachloropropane; the structural formula of the Bergenin derivative is the same as that in Example 1.

[0049] The preparation method of the above-mentioned Bergenia lactone derivatives is as follows:

[0050] (1) Bergenin, 4-chlorobenzaldehyde, potassium carbonate, and DMF were added in a ratio of 2 mmol:4 mmol:6 mmol:25 mL. The mixture was stirred at room temperature for 12 h, then quenched with 6 mol / L hydrochloric acid. The reaction solution was extracted with ethyl acetate, and the ethyl acetate phase was washed with distilled water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain intermediate 1. 1 HNMR and MS (ESI) m / z are consistent with those of Example 1.

[0051] (2) According to the ratio of intermediate 1, baicalin, dimethylamine, and methanol of 2 mmol:4 mmol:6 mmol:60 mL, intermediate 1 from step (1) was added to methanol, followed by the sequential addition of baicalin and a 40% dimethylamine solution. The reaction was carried out at 30 °C, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the solid product was obtained by vacuum filtration, washed with a small amount of methanol, and then dried in a vacuum oven to obtain intermediate 2. The intermediate 2... 1 HNMR and MS (ESI) m / z are consistent with those of Example 1.

[0052] (3) Following the ratio of intermediate 2, bromopropane, and ethanol in step (2) of 1 mmol: 2 mmol: 30 mL, intermediate 2 and bromopropane from step (2) were added to ethanol and refluxed for 50 h. The solid product was obtained by filtration and recrystallization with ethanol to obtain the Bergenin lactone derivative. The Bergenin lactone derivative... 1 The HNMR was consistent with that of Example 1.

[0053] This embodiment also provides a method for preparing the above-mentioned composition containing pyruvate, comprising the following steps:

[0054] Sodium pyruvate, Bergenin lactone derivative, and water for injection are compounded according to the mass percentage, filled into bottles, and then injected with heptachloropropane to obtain the final product.

[0055] Example 3

[0056] This embodiment provides a composition containing pyruvate, which, by mass percentage, consists of the following raw materials: 0.1% sodium pyruvate, 0.04% Bergenin derivative, 10% water for injection, and the balance being tetrafluoroethane; the structural formula of the Bergenin derivative is the same as that in Example 1.

[0057] The preparation method of the above-mentioned Bergenia lactone derivatives is as follows:

[0058] (1) Bergenin, 4-chlorobenzaldehyde, potassium carbonate, and DMF were added in a ratio of 2 mmol:5 mmol:6 mmol:25 mL. The mixture was stirred at room temperature for 16 h, then quenched with 6 mol / L hydrochloric acid. The reaction solution was extracted with ethyl acetate, and the ethyl acetate phase was washed with distilled water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain intermediate 1. 1 HNMR and MS (ESI) m / z are consistent with those of Example 1.

[0059] (2) According to the ratio of intermediate 1, baicalin, dimethylamine, and methanol of 2 mmol: 5 mmol: 8 mmol: 60 mL, intermediate 1 from step (1) was added to methanol, followed by the sequential addition of baicalin and a 40% dimethylamine solution. The reaction was carried out at 70 °C, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the solid product was obtained by vacuum filtration, washed with a small amount of methanol, and then dried in a vacuum oven to obtain intermediate 2. Intermediate 2 1 HNMR and MS (ESI) m / z are consistent with those of Example 1.

[0060] (3) Following the ratio of intermediate 2, bromopropane, and ethanol in step (2) of 1 mmol:3 mmol:50 mL, intermediate 2 and bromopropane from step (2) were added to ethanol and refluxed for 55 h. The solid product was obtained by filtration and recrystallization with ethanol to obtain the Bergenin lactone derivative. The Bergenin lactone derivative... 1 The HNMR was consistent with that of Example 1.

[0061] This embodiment also provides a method for preparing the above-mentioned composition containing pyruvate, comprising the following steps:

[0062] Sodium pyruvate, Bergenin lactone derivative, and water for injection are compounded according to the mass percentage, filled into containers, and then injected with tetrafluoroethane to obtain the final product.

[0063] 2. Comparative Example

[0064] Comparative Example 1

[0065] The difference between Comparative Example 1 and Example 1 is that Bergenin is used instead of Bergenin derivative, while the rest is the same as in Example 1.

[0066] Comparative Example 2

[0067] The difference between Comparative Example 2 and Example 1 is that the Bergenin lactone derivative is omitted, and the amount of sodium pyruvate is increased to 0.1%, while the rest is the same as in Example 1.

[0068] The effects of the drug obtained by the present invention will be described below.

[0069] I. Animal experiments

[0070] Establishment of a rat model of pulmonary fibrosis and administration method

[0071] Experimental animals: 70 SD rats, each weighing 210-220g, were randomly divided into 7 groups with a male-to-female ratio of 1:1. They were acclimatized for 5 days, and one group was randomly selected as the blank control group.

[0072] Model establishment: Rats in each group were anesthetized by intraperitoneal injection of 4% chloral hydrate at a dose of 10 mL / kg. The anesthetized rats were then fixed in a supine position, their necks were shaved, and a longitudinal incision was made to expose the trachea. Except for the blank control group, in all other groups, a syringe was inserted 1 cm into the trachea through the gap between the two tracheal cartilage rings towards the heart. After aspiration without resistance, bleomycin (5 mg / kg) was injected into the trachea. The blank control group received an equal volume of physiological saline. After injection, the rats in each group were rotated upright for 3 minutes to ensure even distribution of the drug in both lungs, and the tissue layers were sutured.

[0073] Administration method: 24 hours after modeling, rats in the blank group and the model group were given an equal amount of physiological saline by gavage. In Examples 1-3 and Comparative Examples 1-2, each group was given 0.6 mg / kg of the preparation (i.e., the amount of sodium pyruvate was 0.6 mg / kg) by nebulization for 28 consecutive days.

[0074] Testing indicators and testing methods:

[0075] (1) Body weight: The body weight of each group of rats was measured on the day of administration and recorded as the initial body weight. The body weight of each group of rats was measured again every 7 days. The experimental results of each group were the average of the experimental results of all rats in each group. The results are shown in Table 1.

[0076] (2) Lung coefficient: Five rats were randomly sacrificed in each group on day 14 and day 28. The lung tissue of each rat was then taken out and weighed to calculate the lung coefficient. Lung coefficient = wet weight of lung (mg) / body weight (mg) × 100%. The experimental results are the average of the experimental results of all rats in each group. The results are shown in Table 2.

[0077] (3) Hydroxyproline content in lung tissue: After 28 days of administration, the remaining 5 rats in each group were sacrificed. The lung coefficient was calculated and the hydroxyproline content in the lung tissue was detected using a hydroxyproline content detection kit. The experimental results are the average of the experimental results of all rats in each group. The results are shown in Table 2.

[0078] Table 1

[0079]

[0080] Table 2

[0081]

[0082] As shown in Table 1, compared with the blank control group, the body weight of rats in the model group decreased significantly on day 21. Compared with the model group, the body weight of rats in Examples 1-3 and Comparative Examples 1-2 increased, with the increase in body weight being more significant in Examples 1-3. Comparative Example 1 used bergapten lactone instead of bergapten derivative, while Comparative Example 2 omitted the bergapten derivative but increased the amount of sodium pyruvate. These results indicate that the composition containing pyruvate obtained in this invention can effectively alleviate the effects of pulmonary fibrosis on rats.

[0083] Table 2 shows that, compared with the blank control group, the lung coefficient and hydroxyproline content in the lung tissue of the model group rats were significantly increased. After administration, compared with the model group, the lung coefficient and hydroxyproline content in the lung tissue of the rats in Examples 1-3 and Comparative Examples 1-2 were decreased, and the decrease in the lung coefficient and hydroxyproline content in the lung tissue of the rats in Examples 1-3 was particularly significant. The above results indicate that the composition containing pyruvate obtained in this invention can effectively improve pulmonary fibrosis in rats. That is, the composition containing pyruvate obtained in this invention can effectively alleviate the progression of pulmonary fibrosis in model rats.

[0084] II. Clinical Trials

[0085] Experimental procedure and administration method

[0086] Experimental subjects: Based on the patients' medical history and relevant clinical treatments, this invention included 25 patients in the acute exacerbation phase of COPD.

[0087] Administration: The composition containing pyruvate obtained in Example 1 was started after the patient was discharged from the hospital, once a day, for a total follow-up period of 6 months.

[0088] Experimental Procedure and Results: Nucleic acid extraction was performed using the TGuide S96 magnetic bead rinsing solution genomic DNA extraction kit. The extract was detected using 16S rRNA detection technology. Primer design: For bacteria, universal bacterial primers 16S were used to amplify the V3+V4 region of the 16S rDNA gene; for fungi, the amplified region was the ITS1 region. Primer designs are shown in Table 3.

[0089] Table 3

[0090]

[0091] The bacterial species and abundance of the present invention were confirmed by comparing with known sequences in the database, and the results are shown in Tables 4-5.

[0092] Table 4 Abundance of phylum-level bacterial communities before and after medication.

[0093]

[0094] Table 5 Abundance of genus-level bacterial communities before and after medication.

[0095]

[0096] As can be seen from Table 4, after using the composition containing pyruvate obtained in this invention, Cynaobacteria (Cyanobacteria) Actinobacteria (Actinomycetes) Firmicutes The abundance of Firmicutes increased. Proteobacteria (Proteobacteria) Bacteroidetes (Bacteroidetes) Fusobacteria The abundance of (Fusobacteria) decreased.

[0097] As can be seen from Table 5, after using the composition containing pyruvate obtained by the present invention, Haemophilus The abundance of (Haemophilus) was significantly reduced, while Streptococcus (Streptococcus) Lactobacillus The abundance of (Lactobacillus) increased.

[0098] In summary, after using the composition containing pyruvate obtained by this invention, the content of Firmicutes significantly increases, including a significant increase in the content of Streptococcus and Lactobacillus genera at the genus level; the content of Proteobacteria significantly decreases, including a significant decrease in the content of Haemophilus genera at the genus level. In conclusion, the composition containing pyruvate obtained by this invention can reduce the abundance of pathogenic bacteria and increase the abundance of probiotics.

[0099] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting 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 shall fall within the scope of protection claimed by the present invention.

Claims

1. A composition comprising pyruvate, characterized in that, The product, by mass percentage, consists of the following raw materials: 0.05-0.1% pyruvate, 0.02-0.04% Bergenin lactone derivative, 5-10% water for injection, and the balance being a propellant; the structural formula of the Bergenin lactone derivative is shown below: ； The pyruvate is sodium pyruvate; the propellant is tetrafluoroethane or heptachloropropane.

2. The composition comprising pyruvate according to claim 1, characterized in that, The preparation method of the Bergenia lactone derivative is as follows: (1) Bergenin was added to DMF, then potassium carbonate and 4-chlorobenzaldehyde were added, stirred at room temperature, and then hydrochloric acid was added to quench the reaction. After purification, intermediate 1 was obtained. (2) Add intermediate 1 from step (1) to methanol, and then add baicalein and dimethylamine solution in sequence to react. After the reaction is completed, purify to obtain intermediate 2. (3) The intermediate 2 and bromopropane from step (2) were added to ethanol and refluxed to obtain Bergenia lactone derivative after purification.

3. The composition comprising pyruvate according to claim 2, characterized in that, In step (1), the molar ratio of Bergenin, 4-chlorobenzaldehyde, 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 intermediate 1, baicalin, 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℃.

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

8. A method for preparing a composition comprising pyruvate according to any one of claims 1-7, characterized in that, Includes the following steps: The pyruvate, Bergenin lactone derivative and water for injection are compounded according to the mass percentage, filled and injected with propellant to obtain the final product.

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