A pyruvate composition for lung tissue regeneration and repair and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a pyruvate composition for lung tissue regeneration and repair and a preparation method thereof. The pyruvate composition comprises the following raw materials in parts by weight: 0.1-0.3 parts of compound I and 110-120 parts of auxiliary materials; the structural formula of the compound I is as follows: The pyruvate composition can promote the repair of lung tissue.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a pyruvate composition for lung tissue regeneration and repair and a preparation method thereof. Background Art

[0002] The lungs are both respiratory organs and filtering organs of the human body. The lungs can secrete mucus to form a protective layer to prevent pathogens or suspended particles in the air from entering the lungs. However, the lungs have limited ability to clear foreign matter, and some foreign matter may penetrate this protective layer and infect or damage the lungs. Clinical studies have found that if the alveolar rupture is small and the number is small, the lung damage is mild, and the patient's own lung function is good, the lungs may repair themselves and heal within a period of time. If the alveolar rupture is serious or a serious infection occurs, it is difficult to repair itself if the patient's own lung function is poor. In this case, targeted treatment is needed to prevent the lungs from continuing to deteriorate.

[0003] However, the current methods of repairing lung damage mainly rely on hormone drugs, drugs that inhibit inflammatory factors, anti-inflammatory drugs and antioxidants, such as dexamethasone, budesonide, fluticasone, etc. The above drugs usually take effect slowly and have great side effects. Based on this, it is urgent to develop a new drug for lung tissue regeneration and repair. Summary of the invention

[0004] The first object of the present invention is to provide a pyruvate composition for lung tissue regeneration and repair, wherein the pyruvate composition can promote the repair of lung tissue.

[0005] The second object of the present invention is to provide a method for preparing a pyruvate composition for lung tissue regeneration and repair, which method is simple and easy to operate.

[0006] The third object of the present invention is to provide the use of the above-mentioned pyruvate composition for lung tissue regeneration and repair in the preparation of a drug for lung tissue repair.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A pyruvate composition for lung tissue regeneration and repair, comprising the following raw materials in parts by weight: 0.1-0.3 parts of compound I, 110-120 parts of auxiliary materials;

[0009] The structural formula of the compound I is as follows:

[0010] .

[0011] Further, the preparation method of compound I is as follows:

[0012]

[0013] (1) mixing a 1-(3,4-diaminophenyl)ethanone solution and a thiosemicarbazide solution, stirring and reacting the mixture, and purifying the reaction solution after the reaction to obtain an intermediate 1;

[0014] (2) Under stirring conditions, adding a sodium pyruvate aqueous solution to a solution of intermediate 1 for reaction, and after the reaction is completed, purifying the reaction solution to obtain intermediate 2;

[0015] (3) mixing the isoliquiritigenin solution and the di(p-nitrobenzene) carbonate solution, adding triethylamine, stirring and reacting, and after the reaction is completed, purifying the reaction solution to obtain intermediate 3;

[0016] (4) Add intermediate 2 to the solution of intermediate 3, then add triethylamine, stir and react. After the reaction is completed, the reaction solution is purified to obtain a pyruvic acid derivative.

[0017] Furthermore, in step (1), the molar ratio of 1-(3,4-diaminophenyl)ethanone to thiosemicarbazide is 1:1-2, and the stirring reaction time is 1-2h.

[0018] Furthermore, in step (2), the molar ratio of sodium pyruvate to intermediate 1 is 1:1-2, the solvent of the solution of intermediate 1 is acetic acid, and the reaction time is 8-10 hours.

[0019] Furthermore, in step (3), the molar ratio of isoliquiritigenin, di(p-nitrobenzene) carbonate and triethylamine is 1:2-3:5-6; the stirring reaction temperature is 40-50°C and the reaction time is 22-26h.

[0020] Furthermore, in step (4), the molar ratio of intermediate 2, intermediate 3 and triethylamine is 1:0.3-0.5:5-6.

[0021] Furthermore, in step (4), the stirring reaction temperature is 20-40° C. and the time is 70-74 hours.

[0022] Furthermore, the auxiliary material is physiological saline.

[0023] A method for preparing a pyruvate composition for lung tissue regeneration and repair comprises compound I and auxiliary materials according to the above-mentioned weight ratio.

[0024] Application of the pyruvate composition for lung tissue regeneration and repair in the preparation of a medicine for lung tissue repair.

[0025] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0026] The present invention provides a new drug for regenerating and repairing lung injury. Based on long-term research and molecular structure design and improvement of chemical drugs, the inventor provides a pyruvate compound (Compound I) with a new structure and efficacy, which belongs to a type of pyruvate derivative. The research results of the present invention show that the prepared pyruvate composition can improve the respiratory function of mice with lung injury; the pyruvate composition can also reduce the content of IL-6 and IL-1β inflammatory factors in the serum and bronchoalveolar lavage fluid of mice with lung injury, thereby reducing lung injury; in addition, the pyruvate composition can also reduce the number of white blood cells and neutrophils in the plasma of mice with lung injury, and reduce the number of neutrophils to reduce lung injury.

[0027] The present invention also provides a method for preparing a pyruvate composition for lung tissue regeneration and repair. The method is simple and easy to operate and can be easily mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the intermediate 1 of Example 1 1 HNMR spectra;

[0029] Figure 2 is the intermediate 2 of Example 1 1 HNMR spectra;

[0030] Figure 3 is the intermediate 3 of Example 1 1 HNMR spectra;

[0031] Figure 4 is the compound Ⅰ in Example 1 1 HNMR spectra;

[0032] Figure 5 This is a graph showing the effect of pyruvate composition on lung resistance in mice with acute injury induced by LPS;

[0033] Figure 6 This is a graph showing the effect of pyruvate composition on respiratory resistance in LPS-induced acute injury mice;

[0034] Figure 7 This is a graph showing the effect of pyruvate composition on dynamic lung compliance in mice with acute injury induced by LPS;

[0035] Figure 8 The figure is a graph showing the effect of pyruvate composition on MPO activity in the lungs of mice with acute injury induced by LPS. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples 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.

[0037] 1. Implementation

[0038] Example 1

[0039] Example 1 provides a pyruvate composition for lung tissue regeneration and repair, the composition comprising the following raw materials in parts by weight: 0.2 parts of compound I, 115 parts of normal saline;

[0040] The structural formula of the compound I is as follows:

[0041] .

[0042] The specific preparation process of the above compound I is as follows:

[0043]

[0044] (1) According to the molar ratio of 1-(3,4-diaminophenyl)ethanone to thiosemicarbazide of 1:1, 1-(3,4-diaminophenyl)ethanone aqueous solution and thiosemicarbazide aqueous solution were mixed, and stirred at room temperature for 1 hour. After the reaction, the reaction solution was filtered, and the precipitate was collected, washed, and freeze-dried to obtain intermediate 1;

[0045] The NMR results of intermediate 1 are:

[0046] 1 H-NMR (400MHz, DMSO)δ=2.34(s,3H),4.96(s,4H),6.75(d,1H),6.89(s,1H),7.12(d,1H),7.77(s,2H),11.31(s,1H). The above results confirmed that the obtained product was the target product.

[0047] (2) Add intermediate 1 (10 mmol) to 20 mL of 1% acetic acid solution to obtain an acetic acid solution of intermediate 1; dissolve sodium pyruvate (10 mmol) in 1.5 mL of deionized water to obtain an aqueous sodium pyruvate solution. Under stirring conditions, add the aqueous sodium pyruvate solution dropwise to the acetic acid solution of intermediate 1 at a molar ratio of 1:1 between sodium pyruvate and intermediate 1, and react at room temperature for 9 hours. After the reaction is completed, filter the reaction solution, collect the precipitate, wash it, and dry it to obtain intermediate 2;

[0048] The NMR results of intermediate 2 are:

[0049] 1 H-NMR (400MHz, DMSO)δ=2.09(s, 3H),2.34(s, 3H),7.61(d, 1H),7.75-7.77(t, 3H),7.86(d, 1H),10.68(s, 1H),11.31(s, 1H). The above results confirmed that the obtained product was the target product.

[0050] (3) According to the molar ratio of isoliquiritigenin, di(p-nitrobenzene) carbonate and triethylamine of 1:2.5:5, a dichloromethane solution of isoliquiritigenin and a dichloromethane solution of di(p-nitrobenzene) carbonate were mixed, and triethylamine was added. The mixture was stirred at 45°C for 24 hours. After the reaction, an appropriate amount of water was added to the reaction solution, and the organic layer was separated and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the organic phase was concentrated and purified by column chromatography to obtain intermediate 3;

[0051] The NMR results of intermediate 3 are:

[0052] 1 H-NMR (400MHz, DMSO)δ=6.82(s,1H),7.10(d,1H),7.31(d,2H),7.54-7.62(m,7H),8.02-8.08(m,2H),8.32(d,4H),16.49(s,1H). The above results confirmed that the obtained product was the target product.

[0053] (4) According to the molar ratio of intermediate 2, intermediate 3 and triethylamine of 1:0.5:5, intermediate 2 was added to the N,N-dimethylformamide solution of intermediate 3, and triethylamine was then added. The mixture was stirred at 30°C for 72 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and appropriate amounts of water and dichloromethane were added. The organic layer was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column to obtain compound I.

[0054] The NMR results of compound Ⅰ are:

[0055] 1 H-NMR (400MHz, DMSO)δ=2.09(s, 6H),2.34(s, 6H),6.82(s, 1H),7.10(d, 1H),7.30(d, 2H),7.61-7.86(m, 9H),8.02-8.08(m, 2H),10.68(s, 2H),11.31(s, 2H),11.48(s, 2H),16.49(s, 1H). The above results confirmed that the product obtained was the target product.

[0056] Example 1 also provides a method for preparing a pyruvate composition for lung tissue regeneration and repair, comprising compound I and physiological saline according to the above weight ratio.

[0057] Example 2

[0058] Example 2 provides a pyruvate composition for lung tissue regeneration and repair, the composition comprising the following raw materials in parts by weight: 0.1 parts of compound I, 110 parts of physiological saline;

[0059] The structural formula of the compound I is as follows:

[0060] .

[0061] The specific preparation process of the above compound I is as follows:

[0062]

[0063] (1) According to the molar ratio of 1-(3,4-diaminophenyl)ethanone to thiosemicarbazide of 1:2, 1-(3,4-diaminophenyl)ethanone aqueous solution and thiosemicarbazide aqueous solution were mixed, and stirred at room temperature for 1.5 hours. After the reaction, the reaction solution was filtered, and the precipitate was collected, washed, and freeze-dried to obtain intermediate 1;

[0064] (2) Add intermediate 1 (10 mmol) to 30 mL of 1% acetic acid solution to obtain an acetic acid solution of intermediate 1; dissolve sodium pyruvate (10 mmol) in 2 mL of deionized water to obtain an aqueous sodium pyruvate solution. Under stirring, add the aqueous sodium pyruvate solution dropwise to the acetic acid solution of intermediate 1 at a molar ratio of sodium pyruvate to intermediate 1 of 1:2, and react at room temperature for 8 hours. After the reaction is completed, filter the reaction solution, collect the precipitate, wash it, and dry it to obtain intermediate 2;

[0065] (3) According to the molar ratio of isoliquiritigenin, di(p-nitrobenzene) carbonate and triethylamine of 1:2:5.5, a dichloromethane solution of isoliquiritigenin and a dichloromethane solution of di(p-nitrobenzene) carbonate were mixed, and triethylamine was added. The mixture was stirred at 50°C for 22 hours. After the reaction, an appropriate amount of water was added to the reaction solution, and the organic layer was separated and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the organic phase was concentrated and purified by column chromatography to obtain intermediate 3.

[0066] (4) According to the molar ratio of intermediate 2, intermediate 3 and triethylamine of 1:0.4:5.5, intermediate 2 was added to the N,N-dimethylformamide solution of intermediate 3, and triethylamine was then added. The mixture was stirred at 40°C for 70 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and appropriate amounts of water and dichloromethane were added. The organic layer was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column to obtain compound I.

[0067] Example 2 also provides a method for preparing a pyruvate composition for lung tissue regeneration and repair, comprising compound I and physiological saline according to the above weight ratio.

[0068] Example 3

[0069] Example 3 provides a pyruvate composition for lung tissue regeneration and repair, the composition comprising the following raw materials in parts by weight: 0.3 parts of compound I and 120 parts of normal saline;

[0070] The structural formula of the compound I is as follows:

[0071] .

[0072] The specific preparation process of the above compound I is as follows:

[0073]

[0074] (1) Mixing an aqueous solution of 1-(3,4-diaminophenyl)ethanone and an aqueous solution of thiosemicarbazide at a molar ratio of 1-(3,4-diaminophenyl)ethanone to thiosemicarbazide of 1:1.5, stirring and reacting at room temperature for 2 h. After the reaction, filtering the reaction solution, collecting the precipitate, washing it, and freeze-drying it to obtain an intermediate 1;

[0075] (2) Add intermediate 1 (10 mmol) to 20 mL of 1% acetic acid solution to obtain an acetic acid solution of intermediate 1; dissolve sodium pyruvate (10 mmol) in 1.5 mL of deionized water to obtain an aqueous sodium pyruvate solution. Under stirring conditions, add the aqueous sodium pyruvate solution dropwise to the acetic acid solution of intermediate 1 at a molar ratio of sodium pyruvate to intermediate 1 of 1:1.5, and react at room temperature for 10 hours. After the reaction is completed, filter the reaction solution, collect the precipitate, wash it, and dry it to obtain intermediate 2;

[0076] (3) According to the molar ratio of isoliquiritigenin, di(p-nitrobenzene) carbonate and triethylamine of 1:3:6, a dichloromethane solution of isoliquiritigenin and a dichloromethane solution of di(p-nitrobenzene) carbonate were mixed, and triethylamine was added. The mixture was stirred at 40°C for 26 hours. After the reaction was completed, an appropriate amount of water was added to the reaction solution, and the organic layer was separated and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the organic phase was concentrated and purified by column chromatography to obtain intermediate 3.

[0077] (4) According to the molar ratio of intermediate 2, intermediate 3 and triethylamine of 1:0.3:6, intermediate 2 was added to the N,N-dimethylformamide solution of intermediate 3, and triethylamine was then added. The mixture was stirred at 20°C for 74 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and appropriate amounts of water and dichloromethane were added. The organic layer was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column to obtain compound I.

[0078] Example 3 also provides a method for preparing a pyruvate composition for lung tissue regeneration and repair, which comprises compound I and physiological saline according to the above-mentioned weight ratio.

[0079] (II) Experimental examples

[0080] 1. Animal grouping, modeling, and drug administration

[0081] A total of 60 SPF healthy BALB / c mice, weighing 18-22g, male, were randomly divided into 6 groups and adaptively fed for 2 days, one of which was a blank control group. The mice in each group were fasted for 12 hours before modeling, and then 0.4% sodium pentobarbital was used to anesthetize the mice at a dose of 0.12mL / 10g. After anesthesia, the mouse mouth was opened with a mouse mouth opener, and lipopolysaccharide (LPS, dose of 4mg / kg) solution was injected into the lungs using a lung liquid quantitative nebulizer, and the model was established.

[0082] Blank control group and model group: given an equal amount of normal saline;

[0083] Experimental group: 2h before modeling, 6h after modeling, and 12h after modeling, the pyruvate composition of Example 1-3 was administered by atomization, and the dose of compound I was 6 mg / kg;

[0084] Positive drug group: Dexamethasone was given at a dose of 6 mg / kg 6 h after modeling, and an equal amount of normal saline was given at other administration time points.

[0085] 2. Detection indicators

[0086] 2.1 24 hours after modeling, whole blood was collected from the eyeballs of mice in each group, and the white blood cells and neutrophils in the whole blood were detected using a blood routine monitor. The results are shown in Table 1.

[0087] 2.2 24 h after modeling, mice were anesthetized with 0.4% sodium pentobarbital at a dose of 0.12 mL / 10 g. After anesthesia, endotracheal intubation was performed and the mice were connected to a small animal pulmonary function analyzer to detect the pulmonary resistance (RL), respiratory resistance (Re), and dynamic pulmonary compliance (Cydn) of each group of mice. The final data were the average values ​​of each group of mice. The results are shown in Figure 5-7 .

[0088] 2.3 24 hours after modeling, whole blood was collected from the eyeballs of mice in each group, and the ELISA kit was used to detect the levels of IL-6 and IL-1β inflammatory factors in the serum. The final data was the average value of each group of mice, and the results are shown in Table 2. The mice were killed immediately after blood collection, and the alveolar lavage fluid of each group of mice was collected. The ELISA kit was used to detect the levels of IL-6 and IL-1β inflammatory factors in the alveolar lavage fluid. The final data was the average value of each group of mice, and the results are shown in Table 3.

[0089] 2.4 The lungs of the mice sacrificed in each group were washed with ice-cold saline, the surface moisture was dried with filter paper, and the MPO activity in the lungs was detected after homogenization with a grinder. The final data were the average values ​​of each group of mice. The results are shown in Figure 8 .

[0090] 3. Test results

[0091] 3.1 Effects of pyruvate combination on blood routine in mice with LPS-induced acute injury

[0092] Table 1

[0093]

[0094] It can be seen from Table 1 that the number of leukocytes and neutrophils in the model group increased significantly compared with the blank control group. Compared with the model group, the number of leukocytes and neutrophils in Example 1-3 groups decreased significantly. Compared with the positive drug group, the number of leukocytes in Example 1-3 groups decreased, and the number of neutrophils decreased significantly. In summary, the pyruvate composition of the present invention can reduce the number of leukocytes and neutrophils in mice with lung injury, thereby alleviating lung injury.

[0095] 3.2 Effects of pyruvate combination on respiratory function in mice with acute LPS-induced injury

[0096] Figure 5 This figure shows the effect of pyruvate composition on lung resistance in mice with acute injury induced by LPS. Figure 6 This figure shows the effect of pyruvate composition on respiratory resistance in mice with acute injury induced by LPS. Figure 7 The figure shows the effect of pyruvate composition on dynamic lung compliance of mice with acute injury induced by LPS. Figure 5-7It can be seen that compared with the blank control group, the lung resistance and respiratory resistance of the model group increased significantly, and the dynamic lung compliance decreased significantly. Compared with the model group, the lung resistance and respiratory resistance of the Example 1-3 groups decreased significantly, and the dynamic lung compliance increased significantly, and there was no significant difference between the Example 1-3 groups and the positive drug group. In summary, the pyruvate composition of the present invention can improve the respiratory function of mice with lung injury.

[0097] 3.3 Effects of pyruvate combination on the levels of IL-6 and IL-1β inflammatory factors in serum and bronchoalveolar lavage fluid of mice with acute injury induced by LPS

[0098] Table 2 Serum levels of IL-6 and IL-1β inflammatory factors

[0099]

[0100] Table 3 Contents of IL-6 and IL-1β inflammatory factors in bronchoalveolar lavage fluid

[0101]

[0102] It can be seen from Table 2-3 that the content of IL-6 and IL-1β inflammatory factors in the serum and alveolar lavage fluid of the model group was significantly increased compared with the blank control group. Compared with the model group, the content of IL-6 and IL-1β inflammatory factors in the serum and alveolar lavage fluid of Example 1-3 groups was significantly reduced. And the content of IL-6 and IL-1β inflammatory factors in the serum and alveolar lavage fluid of Example 1-3 groups was lower than that of the positive drug group. In summary, the pyruvate composition of the present invention can reduce the level of IL-6 and IL-1β inflammatory factors in serum and alveolar lavage fluid. That is, the pyruvate composition of the present invention can inhibit the inflammatory response of lung tissue, thereby reducing the possibility of further damage to the individual's lungs due to inflammation.

[0103] 3.4 Effects of pyruvate combination on MPO activity in the lungs of mice with acute LPS-induced injury

[0104] Figure 8 The figure shows the effect of pyruvate composition on MPO activity in the lungs of mice with acute injury induced by LPS. Figure 8 It can be seen that the MPO activity in the lungs of the mice in the model group was significantly increased compared with the blank control group. The MPO activity in the lungs of the mice in the Example 1-3 group and the positive drug group was significantly reduced compared with the model group, and there was no significant difference between the Example 1-3 group and the positive drug group.

[0105] In summary, the pyruvate composition of the present invention can effectively inhibit the inflammatory response of lung tissue, improve respiratory function, and promote the regeneration and repair ability of lung tissue.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A pyruvate composition for lung tissue regeneration and repair, characterized in that: The method is composed of the following raw materials in parts by weight: 0.1-0.3 parts of compound I and 110-120 parts of auxiliary materials; The structural formula of the compound I is as follows: 。 2. The pyruvate composition for lung tissue regeneration and repair according to claim 1, characterized in that: The preparation method of compound I is as follows: (1) mixing a 1-(3,4-diaminophenyl)ethanone solution and a thiosemicarbazide solution, stirring and reacting the mixture, and purifying the reaction solution after the reaction to obtain an intermediate 1; (2) Under stirring conditions, adding a sodium pyruvate aqueous solution to a solution of intermediate 1 for reaction, and after the reaction is completed, purifying the reaction solution to obtain intermediate 2; (3) mixing the isoliquiritigenin solution and the di(p-nitrobenzene) carbonate solution, adding triethylamine, stirring and reacting, and after the reaction is completed, purifying the reaction solution to obtain intermediate 3; (4) Add intermediate 2 to the solution of intermediate 3, then add triethylamine, stir and react. After the reaction is completed, the reaction solution is purified to obtain the product.

3. The pyruvate composition for lung tissue regeneration and repair according to claim 2, characterized in that: In step (1), the molar ratio of 1-(3,4-diaminophenyl)ethanone to thiosemicarbazide is 1:1-2, and the stirring reaction time is 1-2h.

4. The pyruvate composition for lung tissue regeneration and repair according to claim 2, characterized in that: In step (2), the molar ratio of sodium pyruvate to intermediate 1 is 1:1-2, the solvent of the solution of intermediate 1 is acetic acid, and the reaction time is 8-10 hours.

5. The pyruvate composition for lung tissue regeneration and repair according to claim 2, characterized in that: In step (3), the molar ratio of isoliquiritigenin, di(p-nitrobenzene) carbonate and triethylamine is 1:2-3:5-6; the stirring reaction temperature is 40-50°C and the reaction time is 22-26h.

6. The pyruvate composition for lung tissue regeneration and repair according to claim 2, characterized in that: The molar ratio of intermediate 2, intermediate 3 and triethylamine in step (4) is 1:0.3-0.5:5-6.

7. The pyruvate composition for lung tissue regeneration and repair according to claim 2, characterized in that: The stirring reaction temperature in step (4) is 20-40°C and the time is 70-74h.

8. The pyruvate composition for lung tissue regeneration and repair according to claim 1, characterized in that: The auxiliary material is physiological saline.

9. The method for preparing the pyruvate composition for lung tissue regeneration and repair according to claim 1, characterized in that: Compound I and auxiliary materials can be compounded according to the above weight ratio.

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