Anti-inflammatory composition containing limonin, pneumonia spray and preparation method

Abstract

The invention discloses an anti-inflammatory composition containing limonin, a pneumonia spray and a preparation method. The anti-inflammatory composition containing limonin comprises the following components in parts by weight: 60-70 parts of limonin coated by cyclodextrin and 30-40 parts of clindamycin phosphate. The cyclodextrin is of an annular structure, and a cavity is formed in the cyclodextrin; the outer edge is hydrophilic and the inner cavity is hydrophobic, so that limonin can be wrapped; after the anti-inflammatory composition enters the lung through the respiratory tract, limonin in the anti-inflammatory composition can be gradually released along with dissolution of cyclodextrin; the clindamycin phosphate and the cyclodextrin are both dissolved in water, the clindamycin phosphate shows antibacterial activity after entering a human body, limonin is gradually released along with dissolution of the cyclodextrin, the synergistic effect of the clindamycin phosphate and the limonin can be stable and continuous, then the anti-inflammatory effect is continuous, and it is ensured that the pneumonia treatment effect is continuously and stably carried out.

Description

Technical Field

[0001] The invention relates to the technical field of application of limonin in treating pneumonia, and in particular to an anti-inflammatory composition containing limonin, a pneumonia spray and a preparation method. Background Art

[0002] Pneumonia refers to a lung infection caused by pathogens such as bacteria and viruses, often with typical symptoms such as fever, cough, and sputum. Some pneumonia viruses can be transmitted through the respiratory tract or droplets.

[0003] Currently, the treatment for pneumonia mainly relies on oral medications, which need to be decomposed by the gastrointestinal tract, and their active ingredients need to undergo complex decomposition and extraction in the human body before they can indirectly act on the lungs. This undoubtedly increases the treatment cycle of pneumonia. Since they need to pass through the digestive system, the therapeutic effect of oral medications is greatly reduced, which is not conducive to the rapid recovery of pneumonia.

[0004] Therefore, there is an urgent need to develop drug preparations that can directly act on the lungs and quickly treat pneumonia. Summary of the invention

[0005] The present invention aims to solve one of the problems in the related art at least to a certain extent. To this end, the object of the present invention is to provide an anti-inflammatory composition comprising limonin, wherein the limonin is encapsulated in cyclodextrin, and the cyclodextrin can be used as a sustained-release agent, so that the limonin can effectively enter the lungs and synergize with clindamycin phosphate to achieve rapid treatment of pneumonia.

[0006] An anti-inflammatory composition containing limonin comprises the following components in parts by weight: 60-70 parts of limonin coated with cyclodextrin and 30-40 parts of clindamycin phosphate.

[0007] Furthermore, the molar ratio of the limonin to the cyclodextrin is 1:1-3:1.

[0008] A pneumonia spray comprising the anti-inflammatory composition containing limonin as described above.

[0009] Furthermore, the composition comprises the following components in parts by weight: 15-20 parts of anti-inflammatory composition, 5-10 parts of moisturizing agent, 3-5 parts of pH adjusting agent, and 65-74 parts of pure water.

[0010] Furthermore, the moisturizing agent includes one or more of glycerin, propylene glycol and sorbitol.

[0011] Furthermore, the pH adjuster is sodium citrate or hydrochloric acid buffer solution.

[0012] A method for preparing a pneumonia spray, comprising:

[0013] Cyclodextrin was used to encapsulate limonin;

[0014] Mixing 60-70 parts of cyclodextrin-coated limonin and 30-40 parts of clindamycin phosphate to obtain an anti-inflammatory composition;

[0015] Dissolve 15-20 parts of the anti-inflammatory composition and 5-10 parts of the moisturizer in 65-74 parts of pure water;

[0016] Add 3-5 parts of pH adjuster to adjust the pH value to 5.5-8;

[0017] Fill and add propellant to obtain pneumonia spray.

[0018] Furthermore, the propellant is dichlorodifluoromethane.

[0019] Furthermore, the limonin is encapsulated inside the cyclodextrin by ultrasonic method, saturated aqueous solution method or grinding method.

[0020] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0021] Cyclodextrin is a ring structure with a cavity inside. It has the characteristics of being hydrophilic on the outer edge and hydrophobic on the inner cavity, so it can encapsulate limonin. At the same time, because its outer edge is hydrophilic, the cyclodextrin after encapsulating limonin can dissolve in water, making it easier for limonin to be quickly absorbed by the human body.

[0022] When the anti-inflammatory composition enters the lungs through the respiratory tract, the limonin inside the cyclodextrin will be gradually released as it dissolves; the anti-inflammatory composition of the present application also includes clindamycin phosphate, which is rapidly hydrolyzed into clindamycin after entering the lungs and has an antibacterial effect; through the synergistic effect of limonin and clindamycin, pneumonia can be effectively treated;

[0023] In the present application, clindamycin phosphate and cyclodextrin are both soluble in water, and clindamycin phosphate will show antibacterial activity after entering the human body. Limonin is gradually released as cyclodextrin dissolves. The synergistic effect of the two can be stable and continuous, thereby making the anti-inflammatory effect sustained, ensuring that the treatment effect of pneumonia is sustained and stable. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of this application are all commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0025] The present application provides an anti-inflammatory composition containing limonin, comprising the following components in parts by weight: 60-70 parts of limonin coated with cyclodextrin and 30-40 parts of clindamycin phosphate, wherein the molar ratio of limonin to cyclodextrin is 1:1-3:1.

[0026] Cyclodextrin is a ring-shaped structure with a cavity inside. It has the characteristics of being hydrophilic on the outer edge and hydrophobic on the inner cavity, so it can wrap limonin. At the same time, due to its hydrophilic outer edge, it can be soluble in water, and after entering the lungs through the respiratory tract, it can gradually dissolve, allowing the limonin to be released.

[0027] After entering the lungs, clindamycin phosphate is rapidly hydrolyzed into clindamycin, which has an antibacterial effect; through the synergistic effect of limonin and clindamycin, pneumonia can be effectively treated.

[0028] In the present application, limonin can be encapsulated inside cyclodextrin by ultrasonic method, saturated aqueous solution method or grinding method.

[0029] The working principle of the anti-inflammatory composition in this application is analyzed as follows: cyclodextrin is a ring structure, and a chamber is formed inside it; it has the characteristics of hydrophilic outer edge and hydrophobic inner cavity, so limonin can be wrapped; at the same time, due to its hydrophilic outer edge, the cyclodextrin after wrapping limonin can be soluble in water, which is convenient for limonin to be quickly absorbed by the human body. After the anti-inflammatory composition enters the lungs through the respiratory tract, as the cyclodextrin dissolves, the limonin inside it will be gradually released; the anti-inflammatory composition of this application also includes clindamycin phosphate, which is rapidly hydrolyzed into clindamycin after entering the lungs, and has an antibacterial effect; through the synergistic effect of limonin and clindamycin, pneumonia can be effectively treated. In this application, clindamycin phosphate and cyclodextrin are both soluble in water, and clindamycin phosphate will show antibacterial activity after entering the human body, and limonin will be gradually released as the cyclodextrin dissolves, and the synergistic effect of the two can be stable and continuous, thereby making the anti-inflammatory effect continuous, ensuring that the treatment effect of pneumonia is continued and stable.

[0030] The present application also provides a pneumonia spray, comprising the following components in parts by weight: 15-20 parts of the anti-inflammatory composition as described above, 5-10 parts of a moisturizer, 3-5 parts of a pH regulator, and 65-74 parts of pure water.

[0031] The pH regulator is sodium citrate or hydrochloric acid buffer solution, and the pH regulator stabilizes the pH of the pneumonia spray between 5.5 and 8. The hydrochloric acid buffer solution also has a certain bactericidal effect, which can ensure that the anti-inflammatory composition of the present application can fully exert its efficacy.

[0032] The moisturizer includes one or more of glycerol, propylene glycol and sorbitol. The moisturizer added in the present application can ensure that the anti-inflammatory composition quickly enters the lungs after passing through the respiratory tract. The moisturizer plays a lubricating role to prevent the anti-inflammatory composition from adhering to the nasal cavity or respiratory tract and being unable to move.

[0033] The present application also provides a method for preparing a pneumonia spray, comprising:

[0034] S1: Limonin was coated with cyclodextrin.

[0035] Specifically, limonin can be encapsulated inside cyclodextrin by ultrasonic method, saturated aqueous solution method or grinding method.

[0036] For example, the grinding method is as follows:

[0037] Weigh cyclodextrin or a cyclodextrin derivative, add an appropriate amount of distilled water to grind the cyclodextrin, slowly add limonin in portions under grinding conditions, grind thoroughly, pre-freeze the obtained paste in a -80°C refrigerator for 4 hours, and then use a freeze dryer to dry it at -80°C for 24 hours to obtain cyclodextrin including limonin; wherein the molar ratio of limonin to cyclodextrin or a cyclodextrin derivative is 1:1-3:1.

[0038] S2: mixing 60-70 parts of cyclodextrin-coated limonin and 30-40 parts of clindamycin phosphate to obtain an anti-inflammatory composition;

[0039] S3: dissolving 15-20 parts of the anti-inflammatory composition and 5-10 parts of the moisturizer in 65-74 parts of pure water;

[0040] S4: adding 3-5 parts of pH adjuster to adjust the pH value to 5.5-8;

[0041] S5: Filling and adding propellant to obtain pneumonia spray. The propellant is dichlorodifluoromethane.

[0042] Example 1

[0043] The present embodiment provides an anti-inflammatory composition containing limonin, comprising the following components in parts by weight: 60 parts of limonin coated with cyclodextrin and 40 parts of clindamycin phosphate.

[0044] Example 2

[0045] The present embodiment provides an anti-inflammatory composition containing limonin, comprising the following components in parts by weight: 70 parts of limonin coated with cyclodextrin and 30 parts of clindamycin phosphate.

[0046] Example 3

[0047] The present embodiment provides an anti-inflammatory composition containing limonin, comprising the following components in parts by weight: 68 parts of limonin coated with cyclodextrin and 32 parts of clindamycin phosphate.

[0048] Comparative Example 1

[0049] A sustained-release composition comprises the following components in parts by weight: 68 parts of limonin and 32 parts of clindamycin phosphate.

[0050] Comparative Example 2

[0051] A sustained-release composition comprises the following components in parts by weight: 68 parts of cyclodextrin and 32 parts of clindamycin phosphate.

[0052] Experimental Example 1

[0053] Evaluation of inflammation reduction efficacy (neutrophils)

[0054] 3dpf transgenic neutrophil green fluorescent MPX strain zebrafish were randomly selected in a multi-well plate, and 30 zebrafish were treated in each well (experimental group). The positive control group, normal control group, model control group, experimental group 1, test group 2, experimental group 3, and control group 1 were given water-soluble solution respectively. The normal control group was zebrafish without inflammation and was not treated in any way. Experimental group 1 was the anti-inflammatory composition in Example 1, and copper sulfate pentahydrate was given; experimental group 2 was the anti-inflammatory composition in Example 2, and copper sulfate pentahydrate was given; experimental group 3 was the anti-inflammatory composition in Example 3, and copper sulfate pentahydrate was given. Control group 1 was the anti-inflammatory composition in comparative example 1, and copper sulfate pentahydrate was given; control group 2 was the anti-inflammatory composition in comparative example 2, and copper sulfate pentahydrate was given. The model group was copper sulfate pentahydrate solution.

[0055] The purpose of copper sulfate pentahydrate is to visualize neutrophils.

[0056] Among them, zebrafish were raised in fish farming water at 28°C (water quality: 200 mg instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO 3 ).

[0057] The positive control indomethacin concentration was 60.0 μM, and a normal control group and a model control group were set up at the same time, with a capacity of 3 mL per well. After treatment at 28°C for 1 hour, except for the normal control group, the other experimental groups were given water-soluble copper sulfate pentahydrate to establish a zebrafish inflammation model. After further treatment at 28°C for 2 hours, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software, and the number of neutrophils in the inflammatory site of zebrafish was analyzed. The statistical analysis results of this indicator were used to evaluate the anti-inflammatory efficacy of the sample. The statistical processing results are expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The test results are shown in Table 1.

[0058] Table 1 Evaluation of inflammation-relief efficacy-neutrophil test results (n=10)

[0059] Group The number of neutrophils in the inflammatory area (mean±SE) Normal control group 4.2±0.389*** Model control group 30.5±1.75 Indomethacin 20.1±1.33*** Experimental Group 1 7.25±0.692*** Experimental Group 2 7.54±0.785*** Experimental Group 3 6.24±0.578* Comparison group 1 14.85±0.575*** Comparison group 2 21.5±0.618***

[0060] Compared with the model control group, *p<0.05, ***p<0.001

[0061] Combined with Table 1, it can be seen that the anti-inflammatory effects of experimental group 1, experimental group 2, and experimental group 3 are better than those of the model control group and the positive control group; at the same time, the anti-inflammatory effect of comparison group 1 is not as good as that of experimental group 1, experimental group 2, and experimental group 3, but it is slightly better than that of comparison group 2, because limonin has a certain anti-inflammatory effect. Comparison group 2 does not contain limonin, so its anti-inflammatory effect is worse than that of the positive control group.

[0062] In the present application, limonin is gradually released as cyclodextrin dissolves, and clindamycin phosphate has an antibacterial effect; the synergistic effect between the two can make the anti-inflammatory effect last, thereby achieving a better anti-inflammatory effect. The above-mentioned better anti-inflammatory effect cannot be achieved by simply combining cyclodextrin and clindamycin phosphate or limonin and clindamycin phosphate.

[0063] Example 4

[0064] The pneumonia spray provided in this embodiment includes the following components in parts by weight: 15 parts of the anti-inflammatory composition described above, 10 parts of a moisturizer, 3 parts of a pH regulator, and 72 parts of pure water.

[0065] This embodiment provides a method for preparing a pneumonia spray, comprising:

[0066] S1: Limonin is encapsulated inside cyclodextrin by ultrasonic grinding. The molar ratio of limonin to cyclodextrin is 1:1.

[0067] S2: mixing 68 parts of cyclodextrin-coated limonin and 32 parts of clindamycin phosphate to obtain an anti-inflammatory composition;

[0068] S3: dissolving 15 parts of the anti-inflammatory composition and 10 parts of the moisturizer in 72 parts of pure water;

[0069] S4: add 3 parts of hydrochloric acid buffer solution to adjust the pH value to 5.5-8;

[0070] S5: Fill and add dichlorodifluoromethane as a propellant to obtain a pneumonia spray.

[0071] Example 5

[0072] The pneumonia spray provided in this embodiment includes the following components in parts by weight: 20 parts of the anti-inflammatory composition described above, 5 parts of a moisturizer, 5 parts of a pH regulator, and 70 parts of pure water.

[0073] This embodiment provides a method for preparing a pneumonia spray, comprising:

[0074] S1: Limonin is encapsulated inside cyclodextrin by ultrasonic grinding. The molar ratio of limonin to cyclodextrin is 1:1.

[0075] S2: mixing 68 parts of cyclodextrin-coated limonin and 32 parts of clindamycin phosphate to obtain an anti-inflammatory composition;

[0076] S3: dissolving 20 parts of the anti-inflammatory composition and 5 parts of the moisturizer in 70 parts of pure water;

[0077] S4: add 5 parts of hydrochloric acid buffer solution to adjust the pH value to 5.5-8;

[0078] S5: Fill and add dichlorodifluoromethane as a propellant to obtain a pneumonia spray.

[0079] Example 6

[0080] The pneumonia spray provided in this embodiment includes the following components in parts by weight: 14 parts of the anti-inflammatory composition described above, 8 parts of a moisturizer, 4 parts of a pH regulator, and 74 parts of pure water.

[0081] This embodiment provides a method for preparing a pneumonia spray, comprising:

[0082] S1: Limonin is encapsulated inside cyclodextrin by ultrasonic grinding. The molar ratio of limonin to cyclodextrin is 1:1.

[0083] S2: mixing 68 parts of cyclodextrin-coated limonin and 32 parts of clindamycin phosphate to obtain an anti-inflammatory composition;

[0084] S3: dissolving 14 parts of the anti-inflammatory composition and 8 parts of the moisturizer in 74 parts of pure water;

[0085] S4: add 3 parts of hydrochloric acid buffer solution to adjust the pH value to 5.5-8;

[0086] S5: Fill and add dichlorodifluoromethane as a propellant to obtain a pneumonia spray.

[0087] Comparative Example 3

[0088] The difference from Example 4 is that the anti-inflammatory composition in Example 4 is replaced by an equal amount of pure water.

[0089] Experimental Example 2

[0090] In vivo pneumonia model caused by Streptococcus pneumoniae: SPF-grade SD male rats (weighing about 200 g) were randomly divided into 7 groups, 8 rats in each group, and raised under standard conditions for 7 days, including blank control group, model group, cefuroxime axetil positive control group, experimental group 4, experimental group 5, experimental group 6 and control group 3.

[0091] Before the experiment, the rats were lightly anesthetized with ether inhalation, and then the nasal inhalation method was used. Except for the blank control group rats nasally dripping physiological saline, the model group, the positive control group of cefuroxime axetil tablets, experimental group 4, experimental group 5, experimental group 6 and comparative group 3 were respectively given 0.5mL / kg bacterial solution (concentration of 1.0×109CFU / mL), and the above bacterial solution was slowly dripped into the rat nasal cavity with a syringe needle, and the dripping speed was about 0.05mL / min. After the modeling was completed, at 12h and 24h, the blank control group and the model group mice were given the same dose of physiological saline gavage as the drug group, the positive control group was given cefuroxime axetil tablets (diluted to the same concentration as the spray in Example 6), and the pneumonia spray prepared in Example 4, Example 5, Example 6 and Comparative Example 3 was sprayed three times. 2 hours after the last administration, the rats were anesthetized with 0.4% sodium pentobarbital solution (10 mL / kg), blood was collected from the abdominal aorta and routine blood test results were tested. The results are shown in Table 2.

[0092] Table 2 The number of blood cells in each experimental group

[0093]

[0094]

[0095] It can be seen that the anti-inflammatory effect of the comparison group 3 in which the anti-inflammatory composition was replaced with an equal amount of pure water was not as good as that of the positive control group. The experimental groups 4-6 could reduce the levels of leukocytes, neutrophils and lymphocytes in the blood, and had better activity against pneumonia caused by Streptococcus pneumoniae than cefuroxime axetil tablets.

[0096] Experimental Example 3

[0097] C57 mice (22-25g) were randomly divided into 7 groups, 8 in each group, and raised for 7 days under standard environment. They were respectively blank group, control group, model group, oseltamivir positive control group, experimental group 4, experimental group 5, experimental group 6 and comparison group 3. Influenza B virus was inoculated on the 1st day. Except for the blank control group mice nasal instillation of normal saline, other groups of mice were infected with influenza B H7N9 virus strain (30u) by nasal instillation. After 24h, oral administration for 4 days of continuous intervention treatment was performed, as follows: the blank group and control group mice were given oral administration of normal saline of the same dose of the spray in Example 6, and the mice in the positive control group were given oral administration of oseltamivir of the same dose and concentration of the spray in Example 6, and the pneumonia spray prepared by Example 4, Example 5, Example 6 and Comparative Example 3 was sprayed three times by experimental group 4, experimental group 5, experimental group 6 and comparison group 3 respectively. Continuous administration for 4 days, mouse weight and death were recorded daily. On the last day, blood was collected from the eyeball and the expression levels of NF-KB, TNF-ɑ, IL-1β, and IL-6 in serum were immediately detected. The results are shown in Table 3.

[0098] Table 3 Serum parameters in each experimental group

[0099]

[0100]

[0101] It can be seen that the anti-inflammatory composition was replaced with an equal amount of pure water in the control group 3, and its anti-inflammatory effect was not as good as that of the positive control group. Experimental groups 4-6 can reduce the expression levels of NF-KB, TNF-ɑ, IL-1β, and IL-6 in serum, and have better anti-influenza B virus pneumonia activity than oseltamivir.

[0102] In summary, the cyclodextrin of the present application is a ring structure, and a chamber is formed inside it; it has the characteristics of hydrophilic outer edge and hydrophobic inner cavity, so limonin can be wrapped; at the same time, due to its hydrophilic outer edge, the cyclodextrin after wrapping limonin can be soluble in water, which is convenient for limonin to be quickly absorbed by the human body. After the anti-inflammatory composition enters the lungs through the respiratory tract, as the cyclodextrin dissolves, the limonin inside it will be gradually released; the anti-inflammatory composition of the present application also includes clindamycin phosphate, which is rapidly hydrolyzed into clindamycin after entering the lungs, and has an antibacterial effect; through the synergistic effect of limonin and clindamycin, pneumonia can be effectively treated. In the present application, clindamycin phosphate and cyclodextrin are both soluble in water, and clindamycin phosphate will show antibacterial activity after entering the human body, and limonin will be gradually released as the cyclodextrin dissolves, and the synergistic effect of the two can be stable and continuous, thereby making the anti-inflammatory effect continuous, ensuring that the treatment effect of pneumonia is continuously and stably carried out.

[0103] It can be understood that the above embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the patent scope of the present invention. It should be pointed out that, for ordinary technicians in this field, the above technical features can be freely combined without departing from the concept of the present invention, and several deformations and improvements can be made, which all belong to the protection scope of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should belong to the coverage of the claims of the present invention.

Claims

1. An anti-inflammatory composition comprising limonin, characterized in that The invention comprises the following components in parts by weight: 60-70 parts of limonin coated with cyclodextrin and 30-40 parts of clindamycin phosphate.

2. The anti-inflammatory composition comprising limonin according to claim 1, characterized in that: The molar ratio of the limonin to the cyclodextrin is 1:1-3:

1.

3. A pneumonia spray, characterized in that: The invention comprises an anti-inflammatory composition comprising limonin as claimed in claim 1.

4. A pneumonia spray according to claim 3, characterized in that: The invention comprises the following components in parts by weight: 15-20 parts of anti-inflammatory composition, 5-10 parts of moisturizing agent, 3-5 parts of pH adjusting agent and 65-74 parts of pure water.

5. A pneumonia spray according to claim 4, characterized in that: The humectant includes one or more of glycerin, propylene glycol and sorbitol.

6. A pneumonia spray according to claim 4, characterized in that: The pH regulator is sodium citrate or hydrochloric acid buffer solution.

7. A method for preparing a pneumonia spray, characterized in that: include: Cyclodextrin was used to encapsulate limonin; Mixing 60-70 parts of cyclodextrin-coated limonin and 30-40 parts of clindamycin phosphate to obtain an anti-inflammatory composition; Dissolve 15-20 parts of the anti-inflammatory composition and 5-10 parts of the moisturizer in 65-74 parts of pure water; Add 3-5 parts of pH adjuster to adjust the pH value to 5.5-8; Fill and add propellant to obtain pneumonia spray.

8. The method for preparing a pneumonia spray according to claim 7, characterized in that: The propellant is dichlorodifluoromethane.

9. The method for preparing a pneumonia spray according to claim 7, characterized in that: Limonin was encapsulated inside cyclodextrin by ultrasonic method, saturated aqueous solution method or grinding method.

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