Atomized liquid composition capable of relieving chronic pharyngitis and atomization device

Abstract

The invention relates to an atomized liquid composition which is prepared from the following components in parts by mass: 1-40 parts of a chrysanthemum concentrated solution, 1-10 parts of a lemon concentrated solution, 0-10 parts of a cooling agent, 0-15 parts of a sweetening agent, 0-10 parts of water, 0-25 parts of glycerol and 0-70 parts of propylene glycol. In addition, the invention further provides an atomization device used in cooperation. The atomized liquid composition disclosed by the invention has a certain cough relieving effect and can be used for improving respiratory tract cell injury and inflammation through aerosol inhalation administration.

Description

Technical Field

[0001] The invention belongs to the field of nebulizers, and in particular relates to an nebulizer liquid composition for relieving chronic pharyngitis and an nebulizer device thereof. Background Art

[0002] In the fast-paced life, bad living habits have caused the incidence of acute and chronic pharyngitis to increase year by year. Acute and chronic pharyngitis, with symptoms such as sore throat and dry cough, reduces the quality of life of patients. In the development of throat clearing and soothing products, traditional Chinese medicine has shown unique advantages.

[0003] Common pharyngitis medicines include Lanqin oral liquid, Jinsuozi throat lozenges, Guilin Xiguashuang, Qinghou Liyan granules, etc. These products are mainly oral liquids, lozenges and granules, which have the problems of large dosage and inconvenience in taking or carrying. Compared with these conventional preparations, atomization inhalation is a direct drug delivery method with the respiratory tract and lungs as target organs. It has the advantages of rapid onset, high local drug concentration, small dosage, convenient application and few systemic adverse reactions. It has become an important treatment for respiratory system-related diseases. The handheld atomization device is small and convenient. It can carry the atomization liquid for treating and relieving pharyngitis. It is very convenient to use. However, due to the complex composition and low extraction efficiency of traditional Chinese medicine, it is easy to produce problems such as core sticking when heated and atomized. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an atomizing liquid composition and an atomizing device thereof, wherein the atomizing liquid is mainly composed of chrysanthemum concentrate, lemon concentrate, cooling agent, sweetener, water, glycerol and propylene glycol. The present invention also provides an atomizing device, including an atomizing system, a power system and a control system, with a power of 3-4.5 watts, wherein the heating core (ceramic core) used in the atomizing device has an open porosity of 54±4% and a water absorption rate of 50±5%.

[0005] The technical solution of the present invention is as follows.

[0006] An atomized liquid composition is prepared from the following components in parts by mass.

[0007] Components Mass fraction Chrysanthemum Concentrate 1-40 Lemon Concentrate 1-10 Cooling agent 0-10 Sweeteners 0-15 water 0-10 Glycerol 0-25 Propylene glycol 0-70

[0008] Furthermore, the atomized liquid composition is prepared from the following components in parts by mass.

[0009]

[0010]

[0011] The atomized liquid composition, wherein the mass ratio of the chrysanthemum concentrate to the lemon concentrate is 3-5:1. Preferably, the mass ratio of the chrysanthemum concentrate to the lemon concentrate is 5:1. In the atomized liquid composition, the mass fraction of the chrysanthemum concentrate and the lemon concentrate is 5-25%; preferably, the mass fraction of the chrysanthemum concentrate and the lemon concentrate is 10%.

[0012] In the atomized liquid composition, the sweetener is selected from one or more of neotame, mogroside, and steviol glycoside. Preferably, the sweetener is neotame.

[0013] The atomized liquid composition, the cooling agent is selected from one or more of menthol, menthone, mint extract, menthyl acetate, N,2,3-trimethyl-2-isopropylbutyramide, and natural menthol. Preferably, the cooling agent is selected from WS-23 (N,2,3-trimethyl-2-isopropylbutyramide) and natural menthol.

[0014] The nebulized liquid composition has a chlorogenic acid content of 0.1 to 1 mg / mL. Preferably, the chlorogenic acid content of the nebulized liquid composition is 0.4 to 0.8 mg / mL. The chlorogenic acid comes from the chrysanthemum extract in the formula.

[0015] The atomized liquid composition further comprises 0-5 parts of a preservative, wherein the preservative is selected from one or more of potassium sorbate, dehydroacetic acid and its sodium salt, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, sodium diacetate, calcium propionate and sodium lactate.

[0016] The atomized liquid composition of the present invention can also include one or more of honeysuckle concentrate, loquat leaf concentrate, licorice concentrate, and mulberry leaf concentrate, and the preparation method is the same as that of chrysanthemum concentrate; more preferably, 0-10 parts of honeysuckle concentrate, 0-10 parts of licorice concentrate, 0-10 parts of loquat leaf concentrate, and 0-10 parts of mulberry leaf concentrate are added.

[0017] The method for preparing the atomized liquid composition comprises the following steps:

[0018] A. Weigh the formulated amount of propylene glycol into a container, heat it to 35°C, add the sweetener, and stir evenly when the temperature rises to 40-50°C; add glycerin and stir evenly at 40-50°C;

[0019] B. Accurately weigh the amount of purified water, add it into the container, and stir evenly at 40-50℃;

[0020] C. Accurately weigh the amount of cooling agent and add it into the container, stirring evenly at 40-50℃;

[0021] D. Mix the chrysanthemum concentrate and lemon concentrate in a weight ratio of 5:1, let it stand, slowly add it to the above container, and stir it evenly; after the mixed liquid cools to room temperature, save it for filling the atomizer.

[0022] In the present invention, the preparation method of the chrysanthemum concentrate is as follows:

[0023] Weigh chrysanthemum, add 10 times the amount of 80% (V / V) ethanol aqueous solution, heat and reflux for extraction for 1 hour, filter, and take the filtrate. Add 8 times the amount of 80% (V / V) ethanol aqueous solution to the residue, heat and reflux for extraction for 1 hour, filter, combine the two filtrates, and concentrate the filtrate under reduced pressure at 42°C to 45°C to contain 0.95 to 1.05 g of crude drug per ml. After the concentrated solution is cooled to room temperature, filter or centrifuge to remove solids, take the clear solution, and dilute it with propylene glycol to obtain a concentrated solution with a crude drug concentration of 0.3 g / mL.

[0024] In the present invention, the preparation method of the lemon concentrate is as follows:

[0025] Weigh lemon medicinal materials, add 10 times the amount of 95% ethanol, heat and reflux to extract for 1 hour, filter, and take the filtrate. Add 8 times the amount of 95% ethanol to the residue, heat and reflux to extract for 1 hour, filter, combine the two filtrates, and concentrate the filtrate under reduced pressure at 42°C to 45°C to contain 0.95 to 1.05 g of crude drug per ml, concentrate and cool to room temperature, add water to contain 0.83 g of crude drug per ml, mix well and let stand for more than 12 hours, filter or centrifuge to remove solids, take the clear liquid, add propylene glycol to dilute, and obtain a concentrated solution with a crude drug concentration of 0.3 g / mL.

[0026] The atomizing device is composed of an atomizing rod 3 and an atomizer 1. Figure 1 The atomizer rod 3 includes a control panel 9, a battery 11, an indicator light 2, an airflow sensor 10, and a USB charging interface 12; the atomizer includes a heating core 7, a liquid storage chamber 5, an airflow channel 6, and a nozzle 4, wherein the heating power of the heating core 7 (ceramic core) is 3-4.5 watts, the open porosity is selected to be 54±4%, and the water absorption rate is selected to be 50±5%. Figure 2 .

[0027] The control panel is used to control the atomization action, the liquid storage chamber is used to store the atomization liquid, the airflow sensor is used to detect the suction action and drive the control panel, the battery is used to ensure stable power supply during the driving process, the heating core is used to heat the atomization, and the indicator light is used to indicate the operation, charging and power status of the atomization device. When running, 4 white lights flash according to the power. When charging, 4 white lights flash according to the power. 4 white lights on means 100% power, 3 white lights on means 75% power, 2 white lights on means 50% power, and 1 white light on means 25% power, indicating that the power is about to run out, please charge in time. The USB charging port is used to charge the battery.

[0028] Working principle of the atomization device: the user inhales into the nozzle, negative pressure is generated in the airflow channel and the air pressure sensing channel, the airflow sensor transmits a signal to the control board, the control board sends a signal to the battery, the battery starts to power the heating core (ceramic core), the heating core heats up to atomize the atomized liquid into aerosol, the aerosol is inhaled into the mouth of the user through the airflow channel, when the user stops inhaling from the nozzle, the negative pressure in the airflow sensing channel disappears, the airflow sensor stops sending signals to the control board, and the control board stops working.

[0029] Unless otherwise noted, the ratio of volume to weight in this invention refers to ml / g, L / kg

[0030] The aerosolized liquid composition of the present invention has a certain antitussive effect and improves respiratory cell damage and inflammation through aerosol inhalation. The aerosolized liquid composition is injected into the liquid storage chamber of the aerosol device for patients to use. It is small and convenient, can be carried around, is easy to use, and will not cause the problem of core sticking during heating and aerosolization.

[0031] Instruction Manual

[0032] Figure 1 Appearance diagram of the atomizer; 1-atomizer, 2-indicator light, 3-atomizer rod;

[0033] Figure 2 The cross-sectional view A of the atomizer is a structural diagram, and B is a schematic diagram of the airflow direction; wherein, 4- nozzle, 5- liquid storage chamber, 6- airflow channel, 7- heating core, 8- air pressure sensing channel, 9- control board, 10- airflow sensor, 11- battery, 12- USB charging port;

[0034] Figure 3 Chromatogram of chlorogenic acid determination, A chromatogram of chlorogenic acid standard, B chromatogram of chrysanthemum extract, C chromatogram of chrysanthemum concentrate, D chromatogram of atomized liquid composition prepared from chrysanthemum concentrate;

[0035] Figure 4 Ceramic core sticking condition, A is 1# sample ceramic core, B is 2# sample ceramic core, C is 4# sample ceramic core Figure 5 Comparison of the ceramic core sticking state before and after the atomizer is puffed, A, C, E are ordinary ceramic cores in the initial state, 100 puffs, and 200 puffs, respectively, B, D, and F are the ceramic cores of the present invention in the initial state, 100 puffs, and 200 puffs, respectively;

[0036] Figure 6 Diagram of the CSE preparation device;

[0037] Figure 7 The protective effect of the nebulized liquid composition on CSE-induced BEAS-2B cell membrane and mitochondrial damage;

[0038] Figure 8 The nebulized liquid composition has a protective effect on CSE-induced ROS damage in BEAS-2B;

[0039] Fig. 9 The protective effect of the nebulized liquid composition on TNF-α-induced BEAS-2B cell membrane and mitochondrial damage;

[0040] Fig.10 The nebulized liquid composition has a protective effect on ROS damage of BEAS-2B caused by TNF-α;

[0041] Fig.11 The protective effects of different concentrations of nebulized liquid compositions on CSE-induced cell membrane and mitochondrial damage;

[0042] Fig.12 The protective effects of different concentrations of nebulized liquid compositions on CSE-induced ROS damage;

[0043] Fig.13 The protective effect of different concentrations of nebulized liquid compositions on TNF-α-induced MH-S cell membrane and mitochondrial damage;

[0044] Fig.14 Protective effects of different concentrations of nebulized liquid compositions on TNF-α-induced ROS damage in MH-S cells

[0045] Fig.15 Aerosol particle size distribution of the atomized liquid composition;

[0046] Fig.16 Pathological images of males in the blank control group; A-Blank group, 4#, male, lung, bronchial epithelium (arrow 1) and alveolar epithelium (arrow 2) showed no obvious abnormalities. HE, ×200; B-Blank group, 4#, male, lung, tracheal epithelium (arrow) showed no obvious abnormalities. HE, ×200; C-Blank group, 4#, male, nasal cavity, respiratory epithelium (arrow) showed no obvious abnormalities. HE, ×200; D-Blank group, 4#, male, nasal cavity, olfactory epithelium (arrow) showed no obvious abnormalities. HE, ×200.

[0047] Fig.17 Pathological images of females in the blank control group; A-blank group, 22#, female, lung, bronchial epithelium (arrow 1) and alveolar epithelium (arrow 2) showed no obvious abnormalities. HE, ×200; B-blank group, 22#, female, lung, tracheal epithelium (arrow) showed no obvious abnormalities. HE, ×200; C-blank group, 22#, female, nasal cavity, respiratory epithelium (arrow) showed no obvious abnormalities. HE, ×200; D-blank group, 22#, female, nasal cavity, olfactory epithelium (arrow) showed no obvious abnormalities. HE, ×200.

[0048] Fig.18 Pathological images of males in the nebulized liquid combination group; A-nebulized liquid combination group, 16#, male, no obvious abnormalities were found in the lung, bronchial epithelium (arrow 1) and alveolar epithelium (arrow 2). HE, ×200; B-nebulized liquid combination group, 16#, male, no obvious abnormalities were found in the lung, tracheal epithelium (arrow). HE, ×200; C-nebulized liquid combination group, 16#, male, nasal cavity, respiratory epithelium (arrow), no obvious abnormalities were found. HE, ×200; D-nebulized liquid combination group, 16#, male, nasal cavity, olfactory epithelium (arrow), no obvious abnormalities were found. HE, ×200;

[0049] Fig.19 Pathological diagram of females in the aerosol liquid composition group;

[0050] A nebulized liquid composition group, 36#, female, lung, bronchial epithelium (arrow 1) and alveolar epithelium (arrow

[0051] 2) No obvious abnormalities were found. HE, ×200; B Nebulized liquid combination group, 36#, female, lung, tracheal epithelium (arrow) No obvious abnormalities were found. HE, ×200; C Nebulized liquid combination group, 36#, female, nasal cavity, respiratory epithelium (arrow) No obvious abnormalities were found. HE, ×200; D Nebulized liquid combination group, 36#, female, nasal cavity, olfactory epithelium (arrow) No obvious abnormalities were found. HE, ×200 DETAILED DESCRIPTION

[0052] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0053] Example 1, atomized liquid composition 1 and preparation method thereof

[0054] The atomized liquid composition 1 is prepared from the following raw materials in different mass fractions.

[0055]

[0056]

[0057] The preparation method of the above-mentioned atomized liquid composition 1 comprises the following steps:

[0058] 1) Accurately weigh 54.8 g of propylene glycol, slowly add it into a container, heat it to 35°C, add 0.1 g of neotame, start timing when the temperature rises to 40°C, and stir at 40-50°C for 10 minutes;

[0059] 2) Accurately weigh 20 grams of glycerol, slowly add it into the container, stir at 40-50°C for 10 minutes;

[0060] 3) Accurately weigh 5 grams of purified water, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0061] 4) Accurately weigh 2 grams of cooling agent WS-23 (N,2,3-trimethyl-2-isopropylbutanamide) and 0.1 grams of natural menthol, add them into the container in sequence, and stir at 40-50°C for 25 minutes;

[0062] 5) Turn off the heater;

[0063] 6) Mix 15 g of chrysanthemum concentrate and 3 g of lemon concentrate in a ratio of 5:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0064] 7) After the mixed solution is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0065] Example 2, atomized liquid composition 2 and preparation method thereof

[0066] The atomized liquid composition 2 is prepared from the following raw materials in the following mass fractions.

[0067] Components Mass / g Chrysanthemum Concentrate 30 Lemon Concentrate 6 Menthol 10 Stevioside 15 water 10 Glycerol 25 Propylene glycol 70

[0068] The preparation method of the above-mentioned atomized liquid composition 2 comprises the following steps:

[0069] 1) Accurately weigh 70 g of propylene glycol and slowly add it into a container. Heat it to 35°C and then add 15 g of stevioside. Start timing when the temperature rises to 40°C. Stir at 40-50°C for 10 minutes.

[0070] 2) Accurately weigh 25 grams of glycerol, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0071] 3) Accurately weigh 10 grams of purified water, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0072] 4) Accurately weigh 10 grams of menthol, a cooling agent, and add it to a container. Stir at 40-50°C for 25 minutes.

[0073] 5) Turn off the heater;

[0074] 6) Mix 30 g of chrysanthemum concentrate and 6 g of lemon concentrate in a ratio of 5:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0075] 7) After the mixed solution is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0076] Example 3, atomized liquid composition 3 and preparation method thereof

[0077] The atomized liquid composition 3 is prepared from the following raw materials in the following mass fractions.

[0078] Components Mass / g Chrysanthemum Concentrate 30 Lemon Concentrate 10 Menthyl acetate 10 Mogroside 15 water 10 Glycerol 25 Propylene glycol 70

[0079] The preparation method of the above-mentioned atomized liquid composition 3 comprises the following steps:

[0080] 1) Accurately weigh 70 grams of propylene glycol, slowly add it into a container, heat it to 35°C, add 15 grams of mogroside, start timing when the temperature rises to 40°C, and stir at 40-50°C for 10 minutes;

[0081] 2) Accurately weigh 25 grams of glycerol, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0082] 3) Accurately weigh 10 grams of purified water, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0083] 4) Accurately weigh 10 grams of menthyl acetate, a cooling agent, and add it to a container. Stir at 40-50° C. for 25 minutes.

[0084] 5) Turn off the heater;

[0085] 6) Mix 30 g of chrysanthemum concentrate and 10 g of lemon concentrate in a ratio of 3:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0086] 7) After the mixed solution is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0087] Example 4, atomized liquid composition 4 and preparation method thereof

[0088] The atomized liquid composition 4 is prepared from the following raw materials in the following mass fractions.

[0089] Components Mass / g Chrysanthemum Concentrate 40 Lemon Concentrate 1 Menthyl acetate 10 Mogroside 15 water 10 Glycerol 25 Propylene glycol 70

[0090] The preparation method of the above-mentioned atomized liquid composition 4 comprises the following steps:

[0091] 1) Accurately weigh 70 grams of propylene glycol, slowly add it into a container, heat it to 35°C, add 15 grams of mogroside, start timing when the temperature rises to 40°C, and stir at 40-50°C for 10 minutes;

[0092] 2) Accurately weigh 25 grams of glycerol, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0093] 3) Accurately weigh 10 grams of purified water, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0094] 4) Accurately weigh 10 grams of menthyl acetate, a cooling agent, and add it to a container. Stir at 40-50° C. for 25 minutes.

[0095] 5) Turn off the heater;

[0096] 6) Mix 40 g of chrysanthemum concentrate and 1 g of lemon concentrate at a ratio of 40:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0097] 7) After the mixed solution is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0098] Example 5, atomized liquid composition 5 and preparation method thereof

[0099] The atomized liquid composition 5 is prepared from the following raw materials in the following mass fractions.

[0100] Components Mass / g Chrysanthemum Concentrate 1 Lemon Concentrate 1 Glycerol 2 Propylene glycol 5

[0101] The preparation method of the above-mentioned atomized liquid composition 5 comprises the following steps:

[0102] 1) Accurately weigh 5 grams of propylene glycol, slowly add it into a container, and heat it to 35°C;

[0103] 2) Accurately weigh 2 grams of glycerol, slowly add it into the container, stir at 40-50°C for 10 minutes;

[0104] 3) Turn off the heater;

[0105] 4) Mix 1 gram of chrysanthemum concentrate and 1 gram of lemon concentrate in a ratio of 1:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0106] 5) After the mixed liquid is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0107] Example 6, atomized liquid composition 6 and preparation method thereof

[0108] The atomized liquid composition 6 is prepared from the following raw materials in the following mass fractions.

[0109] Components Mass / g Chrysanthemum Concentrate 6 Lemon Concentrate 2 Menthone 1 Stevioside 2 water 2 Propylene glycol 50

[0110] The preparation method of the above-mentioned atomized liquid composition 6 comprises the following steps:

[0111] 1) Accurately weigh 50 grams of propylene glycol and slowly add it into a container. Heat it to 35°C and then add 2 grams of stevioside. When the temperature rises to 40°C, start timing and stir at 40-50°C for 10 minutes.

[0112] 2) Accurately weigh 2 grams of purified water, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0113] 3) Accurately weigh 1 gram of the cooling agent menthone and add it to the container, stir at 40-50°C for 25 minutes;

[0114] 4) Turn off the heater;

[0115] 5) Mix 6 g of chrysanthemum concentrate and 2 g of lemon concentrate in a ratio of 3:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0116] 6) After the mixed solution is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0117] Example 7, atomized liquid composition 7 and preparation method thereof

[0118] The atomized liquid composition 7 is prepared from the following raw materials in the following mass fractions.

[0119] Components Mass / g Chrysanthemum Concentrate 12 Lemon Concentrate 3 WS-23 6 Neotame 12 water 10 Glycerol 25

[0120] The preparation method of the above-mentioned atomized liquid composition 7 comprises the following steps:

[0121] 1) Accurately weigh 25 grams of glycerol and slowly add it to a container. Heat it to 35°C and then add 12 grams of neotame. Start timing when the temperature rises to 40°C. Stir at 40-50°C for 10 minutes.

[0122] 2) Accurately weigh 10 grams of purified water, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0123] 3) Accurately weigh 6 grams of cooling agent WS-23 and add it into the container, stir at 40-50°C for 25 minutes;

[0124] 4) Turn off the heater;

[0125] 5) Mix 12 g of chrysanthemum concentrate and 3 g of lemon concentrate in a ratio of 4:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0126] 6) After the mixed solution is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0127] Experimental Example 1: Comparison of Chrysanthemum Extraction Methods (Determination of Chlorogenic Acid Content)

[0128] 1. Experimental materials:

[0129] 1.1 Chinese medicinal materials and reagents:

[0130] Chrysanthemum: Yunnan New Century Chinese Medicine Pieces Co., Ltd.; Lemon: Bozhou Yunyuantang Flower Tea Co., Ltd.; Honeysuckle: Shandong Fengliaoxing Medicinal Pieces Co., Ltd.; Ethanol: Jilin Province Xintianlong Industrial Co., Ltd.; Propylene glycol: CNOOC Shell Petrochemical Co., Ltd.; Neotame: Shandong Benyue Biotechnology Co., Ltd.; Natural menthol: Xiangsheng Flavor (Huai'an) Co., Ltd.; WS-23: Kunshan Yaxiang Flavor Co., Ltd.; Acetic acid: analytical grade, Tianjin Zhiyuan Chemical Reagent Co., Ltd.; Acetonitrile: chromatographic grade, Tianjin Kangkede Technology Co., Ltd.; Chlorogenic acid standard: purity 96.1%, China Food and Drug Inspection Institute.

[0131] 1.2 Instruments and equipment: Rotary evaporator: Tokyo Rika, N-1300D-WB; high-speed refrigerated centrifuge: Hettich, Rotina 380R, Agilent high-performance liquid chromatograph.

[0132] 2. Preparation of chrysanthemum concentrate:

[0133] Accurately weigh chrysanthemum medicinal materials into a container, add 10 times the amount of 30% (V / V) ethanol aqueous solution, reflux extraction for 1 hour, filter, and take the filtrate. Add 8 times the amount of 30% (V / V) ethanol aqueous solution to the residue, heat reflux extraction for 1 hour, filter, and combine the two filtrates.

[0134] The filtrate was concentrated under reduced pressure at 42°C to 45°C to contain 0.65 to 0.69 g of crude drug per ml, and diluted with water to obtain a chrysanthemum extract with a crude drug concentration of 0.3 g / ml.

[0135] 3. Preparation of atomized liquid composition

[0136] 1) Accurately weigh 54.8 g of propylene glycol, slowly add it into a container, heat it to 35°C, add 0.1 g of neotame, start timing when the temperature rises to 40°C, and stir at 40-50°C for 10 minutes;

[0137] 2) Accurately weigh 20 grams of glycerol, slowly add it into the container, stir at 40-50°C for 10 minutes;

[0138] 3) Accurately weigh 5 grams of purified water, slowly add it into the container, and stir at 40-50°C for 10 minutes;

[0139] 4) Accurately weigh 2 grams of cooling agent WS-23 (N,2,3-trimethyl-2-isopropylbutanamide) and 0.1 grams of natural menthol, add them into the container in sequence, and stir at 40-50°C for 25 minutes;

[0140] 5) Turn off the heater;

[0141] 6) Mix 15 g of chrysanthemum concentrate and 3 g of lemon concentrate in a ratio of 5:1, let stand for 1 hour, then slowly add to the container and stir for 5 minutes;

[0142] 7) After the mixed solution is cooled to room temperature (cover and seal), save it for filling the atomizer.

[0143] 4. Determination of chlorogenic acid in the aerosol liquid composition

[0144] 4.1 Preparation of chlorogenic acid standard solution:

[0145] Weigh 0.02 g of chlorogenic acid standard, accurately weigh it, place it in a 10.0 ml volumetric flask, add the mobile phase to dissolve and dilute to the scale, mix well to obtain the chlorogenic acid standard stock solution.

[0146] Accurately measure 1.00 ml of chlorogenic acid standard stock solution into a 10.0 ml volumetric flask, dilute to the scale with mobile phase, and mix well to obtain the chlorogenic acid standard solution.

[0147] 4.2 Sample solution preparation:

[0148] 4.2.1 Preparation of chrysanthemum extract and chrysanthemum concentrate sample solutions:

[0149] Chrysanthemum extract and chrysanthemum concentrate were diluted with mobile phase respectively, passed through 0.45 μm filter membrane, and the filtrate was used for liquid chromatography analysis.

[0150] 4.2.2 Preparation of sample solution of nebulized liquid composition:

[0151] The atomized liquid composition was diluted with the mobile phase, passed through a 0.45 μm filter membrane, and the filtrate was subjected to liquid chromatography analysis.

[0152] 4.3 Preparation of standard curve:

[0153] The chlorogenic acid standard solutions were respectively aspirated, diluted with the mobile phase and made up to the concentrations of 0.002, 0.01, 0.02, 0.04, and 0.08 mg / mL standard series in volumetric flasks.

[0154] 4.4 Liquid chromatography conditions:

[0155] Chromatographic column: ODS C18 column, (250×4.6mm, 5μm)

[0156] The mobile phase is 0.5% acetic acid aqueous solution: acetonitrile = 9:1

[0157] The flow rate is 1.0 ml / min.

[0158] The column temperature was 35°C.

[0159] The detection wavelength is 327nm

[0160] The injection volume is 10 μL

[0161] Chromatographic analysis: inject the standard solution and sample solution into the chromatographic column, determine the qualitative properties by retention time, and determine the quantitative properties by comparing the sample peak area with the standard to calculate the content.

[0162] 5. Measurement results

[0163] The chromatogram under the above chromatographic conditions is shown in Figure 3 , A is a standard chromatogram of chlorogenic acid, B is a chromatogram of chrysanthemum extract, C is a chromatogram of chrysanthemum concentrate, and D is a chromatogram of a nebulized liquid composition prepared from chrysanthemum concentrate. The concentration of chlorogenic acid in chrysanthemum concentrate is 0.94 mg / ml; the concentration of chlorogenic acid in chrysanthemum extract is 0.51 mg / ml.

[0164] The chlorogenic acid concentration in the atomized liquid composition prepared from the chrysanthemum concentrate is 0.13 mg / ml; in the two extraction processes, under the same crude drug concentration, the chlorogenic acid content of the chrysanthemum concentrate is higher than that of the chrysanthemum extract. Experimental Example 2: Screening of plant formulas in atomized liquid compositions

[0165] 1. Experimental Methods

[0166] Medicinal materials such as honeysuckle, emblica fruit, loquat leaf, liquorice, mulberry leaf, tangerine peel, olive, purslane and dandelion are prepared according to the preparation process of the chrysanthemum concentrate of the present invention to obtain the concentrated solutions of the medicinal materials.

[0167] Mix according to the table below to examine the compatibility of chrysanthemum concentrate with lemon, honeysuckle, emblica, loquat leaf, licorice, mulberry leaf, tangerine peel, olive, purslane and dandelion concentrate in the formula.

[0168] Table 1 Ratio of chrysanthemum concentrate and other medicinal materials

[0169]

[0170] 2. Experimental results

[0171] The experimental results show that after mixing the emblica concentrate, dandelion concentrate and chrysanthemum concentrate, the solution becomes turbid, and these two plants are not suitable for use; the remaining concentrates can be mixed with chrysanthemum concentrate and lemon concentrate, especially when lemon concentrate is used together with chrysanthemum concentrate, the atomization device has a unique taste and has the characteristic aroma of chrysanthemum and lemon.

[0172] Experimental Example 3: Screening of component ratios in atomized liquid compositions

[0173] 1. Experimental Methods

[0174] The mixture was mixed according to the following Table 2, and the effects of different added weights and different proportions of chrysanthemum concentrate and lemon concentrate on taste and core sticking were investigated. The atomizing device used was the atomizing device of the present invention, wherein the heating power was set to 4 watts, the open porosity of the heating core was selected to be 54%, and the water absorption rate was selected to be 50%.

[0175] Table 2 Different ratios of chrysanthemum concentrate and lemon concentrate

[0176]

[0177]

[0178] 2. Experimental results

[0179] Adding 20% ​​of the mixture of chrysanthemum concentrate and lemon concentrate will cause core sticking and carbon deposition (see Figure 4 , A is the ceramic core of sample 1#, and the ceramic core of sample 2#); when the addition amount of the mixture of chrysanthemum concentrate and lemon concentrate was 10%, the sticking of the core was improved. The carbon deposition phenomenon of samples 3# and 4# was improved after adding water. The carbon deposition phenomenon of sample 4# was improved best when adding 5% water ( Figure 4 C is the ceramic core of sample 4#).

[0180] During the tasting process, it was found that when the ratio of chrysanthemum concentrate to lemon concentrate was 5:1, the aroma was more natural and elegant, producing the unique characteristic aroma of chrysanthemum and lemon; the carbon deposition phenomenon of 5#-10# samples was improved, but the aroma was not as good as 3# and 4# samples. Therefore, in the subsequent experiments, the ratio of chrysanthemum concentrate to lemon concentrate was set at 5:1.

[0181] Experimental Example 4: Screening of Heating Cores

[0182] The atomized liquid composition of the present invention contains Chinese herbal medicine extract components, and the core will be sticky during the suction process. To avoid the core sticking phenomenon, the heating power of the atomizer of the present invention is set to 3-4.5 watts, the open porosity of the heating core is selected to be 54±4%, and the water absorption rate is selected to be 50±5%.

[0183] 1. Selection of heating power of atomization device

[0184] The heating power of the atomizer is low, and the temperature of the heating core is correspondingly low, so it is not easy to cause the core to burn; at the same time, low power releases less harmful substances and lower content than high power, which is safer. The following table compares the harmful substances and content in the aerosol released by atomizers of different powers for the same atomizing liquid composition. The aerosol was tested by gas phase, and the results are as follows.

[0185] Table 3 Harmful substances test results (μg / 100 mouthfuls)

[0186]

[0187] The data in the table show that at a power of 4 watts, the only harmful substances released in the aerosol are formaldehyde and acetaldehyde, and the content is very low (ND means not detected).

[0188] 2. Screening of heating core parameters of atomization device

[0189] The test results of the density, porosity and water absorption of the ceramic cores of the heating cores used in the atomization device of the present invention (samples 1 to 5) and the heating cores commonly used on the market (samples 6 to 10) are shown in Table 4.

[0190] Table 4 Heating core parameter test

[0191] Sample No. Open porosity (%) Water absorption (%) <![CDATA[Bulk density (g / cm 3 )]]> <![CDATA[Total volume (cm 3 )]]> Sample 1 56.90 50.77 1.117 0.06 Sample 2 51.61 47.76 1.077 0.06 Sample 3 50.88 43.94 1.154 0.06 Sample 4 60.66 54.68 1.013 0.06 Sample 5 51.61 47.06 1.094 0.06 Sample 6 36.00 22.69 1.582 0.08 Sample 7 32.88 19.83 1.653 0.07 Sample 8 38.10 26.45 1.436 0.08 Sample 9 42.17 29.17 1.442 0.08 Sample 10 35.14 21.87 1.617 0.07

[0192] The open porosity of commonly used heating cores (ceramic cores) on the market is 30-40%, and the open porosity of the heating core of the atomizer device of the present invention is 54±4%. While ensuring the same structural stability, the heating core of the atomizer device of the present invention can absorb 37-83% more atomized liquid than the traditional ceramic core, and more effective substances can be released each time the heating and atomization are performed; in addition, more atomized liquid adsorption can maintain the atomized liquid within a relatively low temperature range under the same heating power, thereby protecting the effective substances in the atomized liquid from decomposition and failure due to excessive heat.

[0193] The water absorption rate of commonly used heating cores (ceramic cores) on the market is 20-30%, and the water absorption rate of the heating core of the atomizer device of the present invention is 50±5%, which is 25-67% higher than the water absorption rate of ordinary ceramic cores, indicating that the heating core of the atomizer device of the present invention has a stronger atomization liquid adsorption capacity; therefore, when the atomizer is working, it can ensure that the atomization liquid fully and quickly penetrates into the ceramic core, greatly reducing the risk of core sticking due to dry burning of the ceramic core, thereby ensuring the safety of the product during use.

[0194] Experimental Example 5: Determination of the content of chlorogenic acid in the aerosol atomized by the atomizer

[0195] This experiment established a method for using the atomizing device of the present invention to generate an aerosol by atomizing the atomizing liquid composition, using a Cambridge filter to capture the aerosol, and using high performance liquid chromatography to determine the content of the active ingredient chlorogenic acid in the aerosol. The two atomizers prepared with a ceramic core commonly used on the market and a ceramic core of the present invention and loaded with the atomizing liquid of the present invention were compared. The difference in the content of the active ingredient chlorogenic acid in the aerosol formed after the atomizing liquid was atomized and transferred from the atomizing liquid to the aerosol was found. The results showed that under the same suction conditions, the atomizing device of the present invention can atomize and inhale more effective substances in the atomizing liquid, improve the bioavailability of small molecular substances, and better exert the efficacy of the atomizing liquid.

[0196] 1. Experimental materials:

[0197] 1.1 Atomizer:

[0198] Empty atomizers made of ceramic cores commonly used in the market and the ceramic core selected by the present invention were filled with the atomizing liquid composition of the present invention to prepare two types of atomizers for aerosol testing.

[0199] 1.2 Reagents and drugs:

[0200] Chlorogenic acid standard (China Food and Drug Control Institutes, batch number 110753-202119), methanol (Tianjin Concord Technology Co., Ltd., batch number 2021MH013), acetonitrile (Tianjin Concord Technology Co., Ltd.,

[0201] Batch No. 2022AH006), propylene glycol (McLean, Batch No. C13047247), glycerol (Yongda Chemical Reagent Co., Ltd., Batch No. 20211016), ethanol (Tianjin Zhiyuan Chemical Reagent Co., Ltd., Batch No. 2022401329), phosphoric acid (Tianjin Zhiyuan Chemical Reagent Co., Ltd., Batch No. 2022090142), ultrapure water (homemade)

[0202] 1.3 Instruments and equipment

[0203] High performance liquid chromatograph (1260, Agilent), chromatographic column (Diomansil Plus C18, 4.6*250mm, 5μm Dima), Cambridge filter (Φ44mm, Waterman), suction machine (FH-Y1216, Feihong Instrument), one-tenth balance (SQP Sartorius), one-hundred-thousandth balance (CPA225D, Sartorius), ultrapure water machine (casadal, Pall)

[0204] 2. Experimental methods:

[0205] The content of chlorogenic acid in the aerosolized liquid composition was determined before puffing. After 100 and 200 puffs, the aerosol was captured by a Cambridge filter. The captured aerosol was processed and the content of chlorogenic acid in the aerosol was determined. After puffing, the content of chlorogenic acid in the aerosolized liquid composition was determined again, and the change in the content of chlorogenic acid in the aerosolized liquid composition before and after puffing could be obtained.

[0206] 2.1 Suction parameters

[0207] Table 5 Suction parameters of suction machine

[0208]

[0209] 2.2 Chromatographic conditions

[0210] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the following table: 0-8 min, 14% → 19% A, 8-14 min, 19% A, 14-34 min, 19% → 31% A

[0211] 34-35min, 31%→90%A, 35-40min, 90%A; column temperature: 25℃; flow rate: 0.7ml / min, detection wavelength: 327nm. The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 10,000.

[0212] 2.3 Preparation of reference solution:

[0213] Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, place it in a brown volumetric flask, and add 75% methanol to make a solution containing 0.022 mg per 1 ml.

[0214] 2.4 Preparation of test solution:

[0215] 2.4.1 Chlorogenic acid-solvent:

[0216] Weigh the chlorogenic acid standard, accurately weigh it and place it in a 2 ml volumetric flask, dissolve it in the mixed solution prepared according to the process ratio (mass ratio) in Table 6 (ultrasonication in cold water, shake the volumetric flask while ultrasonicating to ensure that the chlorogenic acid standard is completely dissolved), make up to volume, and shake well.

[0217] Table 6 Solvent process ratio

[0218] name Propylene glycol glycerin water 95% ethanol Proportion 66.3％ 20.42％ 9.19％ 4.08％

[0219] 2.4.2 Chlorogenic acid-nebulizer liquid composition:

[0220] Weigh the chlorogenic acid standard accurately, place it in a 2 ml volumetric flask, dissolve it in the nebulized liquid composition 1 of Example 1 of the present invention (ultrasonication in cold water, shake the volumetric flask while ultrasonicating to ensure that the chlorogenic acid standard is completely dissolved), make up to volume, and shake well.

[0221] 2.5 Sample pretreatment:

[0222] 2.5.1 Treatment of the atomized liquid composition before inhalation:

[0223] Take 1 g of each test solution prepared in 2.4, accurately weigh it, put it in a stoppered conical flask, accurately add 10 ml of 75% methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 minutes (the same as the Cambridge filter ultrasonic treatment), cool it, weigh it again, make up the lost weight with 75% methanol, shake it well, filter it, and take the filtrate.

[0224] 2.5.2 Treatment of Cambridge filter discs:

[0225] Take appropriate amount of the test solution prepared in 2.2.3 in different atomizers, accurately weigh the total weight of the atomizer and the solution, and aspirate according to the above suction conditions for a total of 200 mouthfuls. Replace the filter disc every 100 mouthfuls and take photos to observe the sticking of the ceramic core. Combine the Cambridge filter discs after aspiration, and wipe the inner wall of the front cover of the collector with a new Cambridge filter disc, and put them into the stoppered conical bottle. Accurately add 20ml of 75% methanol, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 75% methanol, shake well, filter, and take the filtrate to obtain the aerosol solution.

[0226] 2.5.3 Treatment of the atomized liquid composition after inhalation:

[0227] Accurately weigh the total weight of the nebulizer and the solution after suction. Open the nebulizer, place the entire nebulizer and the remaining solution in the nebulizer in a suitable container, accurately add 20 ml of 75% methanol, weigh the weight, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 75% methanol, shake well, filter, and take the filtrate.

[0228] 2.6 Determination of chlorogenic acid content:

[0229] Accurately pipette 10 μl of the reference solution in 2.3 and the solution prepared in 2.5 respectively and inject them into the liquid chromatograph.

[0230] 3. Experimental results

[0231] 3.1 Results of determination of chlorogenic acid content in aerosol

[0232] Table 7 Test results of chlorogenic acid content in aerosol

[0233]

[0234] Among them, chlorogenic acid atomization rate % = aerosol chlorogenic acid / total amount of chlorogenic acid before inhalation × 100%

[0235] 3.2 Results of ceramic core paste state

[0236] Comparison of the state of the nebulizer before and after suction, see Figure 5 , where A, C, and E are ordinary ceramic cores in the initial state, 100 puffs of suction, and 200 puffs of suction, respectively, and B, D, and F are the ceramic cores of the present invention in the initial state, 100 puffs of suction, and 200 puffs of suction, respectively. Figure 5It can be seen that the atomized liquid compositions of the two ceramic cores are light yellow before the start of puffing; after 100 puffs, the color of the atomized liquid composition of the ordinary ceramic core begins to darken, and the ceramic core begins to turn black and carbon deposition occurs; the color of the atomized liquid composition of the ceramic core of the present invention becomes slightly darker, but there is no carbon deposition; after 200 puffs, the color of the atomized liquid composition of the ordinary ceramic core becomes darker, and the ceramic core turns black and has serious carbon deposition; the color of the atomized liquid composition of the ceramic core of the present invention becomes darker, but there is no carbon deposition.

[0237] 4. Experimental conclusion:

[0238] In this study, a series of detection methods for chlorogenic acid in aerosols were established, by which the content of chlorogenic acid in aerosols can be detected.

[0239] Two types of atomizers were used in this experiment. From the results of core sticking, it can be seen that the state of the atomizer of the present invention after 100 and 200 puffs was better than that of the atomizer made of ordinary ceramic core, which shows that increasing the open porosity of the ceramic core can improve the core sticking condition of the ceramic core.

[0240] The atomizer core of the present invention has a higher proportion of the total amount of chlorogenic acid in the aerosol / atomized liquid composition, indicating that increasing the porosity of the atomizer core can also increase the atomization rate of chlorogenic acid. Before and after inhalation, the loss percentage of chlorogenic acid in the atomized liquid of the ordinary ceramic core (sample 6) is 35.6-37.9%, and the percentage of chlorogenic acid transferred from the atomized liquid to the aerosol is 0.022-0.024%; the loss percentage of chlorogenic acid in the ceramic core used in the present invention is 59.5-62.4%; the percentage of chlorogenic acid transferred from the atomized liquid to the aerosol is 0.048-0.057%.

[0241] Experimental Example 6: Atomized Liquid Function Evaluation Experiment

[0242] 1. Sample preparation

[0243] (I) Preparation of atomized liquid composition

[0244] According to the formula and method of Example 1, atomized liquid composition 1 was prepared.

[0245] (II) Preparation of Cigarette Smoke Extract (CSE)

[0246] Place 50 cigarettes in a smoke generator, which is connected to a 100mL filter bottle, and the other end of the filter bottle is connected to a circulating water pump. Place 50mL PBS in the bottle, and burn the cigarette at a speed of 2min45s / cigarette. The filter bottle collects cigarette smoke. After the smoke suspension is prepared, seal and shake well, adjust the pH value to 7.4, and filter with a 0.22μm filter membrane. The prepared solution is 100% CSE. CSE preparation device see Figure 6 .

[0247] 2. Experimental Methods

[0248] 1. Mouse cough suppression experiment

[0249] Kunming mice were adaptively fed for 3 days and randomly divided into 5 groups: positive control group, low-dose group, medium-dose group, high-dose group and model group (no aerosol liquid composition was given, only ammonia water was used to make the model), 12 mice in each group. The positive drug group was given dextromethorphan hydrobromide tablets (31.2 mg / kg, 0.1 mL / 10 g) by gavage, and the low-dose group, medium-dose group and high-dose group of the aerosol liquid composition were administered by oral and nasal inhalation: the RTE-CY02 atomizer suction pressure was 1.5 kPa during administration, each suction was 5 s, the suction interval was 5 s, and the two channels were opened at the same time to ensure that the concentration in the cavity was saturated. The mice were fixed in the oral and nasal exposure tower, and the high, medium and low dose groups were given the aerosol liquid composition by aerosol inhalation for 5 min, 10 min and 20 min respectively. During the experiment, the CEL-712 Microdust Pro monitor was used to monitor the aerosol concentration of the nebulized liquid composition for 2 consecutive days. On the third day, 1 hour after the positive drug was administered, the multifunctional cough-inducing and wheezing instrument was used to monitor the aerosol concentration of the nebulized liquid composition.

[0250] After 12 seconds of stimulation with 13% concentrated ammonia solution nebulized at a rate of 1.2 mL / min, the cough latency and coughing within 3 minutes after administration were recorded (coughing was judged by violent contraction of the abdominal muscles and opening of the mouth, when a slight coughing sound could be heard. Two experimenters observed and recorded the cough latency and coughing frequency at the same time).

[0251] 2. Protective effect of the nebulized liquid composition on CSE-induced BEAS-2B cell membrane and mitochondrial damage

[0252] The cells were inoculated in a black transparent bottom 96-well plate. The experiment was set up with 5 groups, including a blank group, a CSE intervention group, a 0.01% nebulized liquid composition group, a 0.05% nebulized liquid composition group, and a 0.1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank control group, 5% CSE was added to the CSE intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 5% CSE were added to the nebulized liquid composition group for co-culture for 24 hours. The BioTek Cytaion5 high-content imager was used to simultaneously stain the nucleus with Hoechst 33342 and the mitochondrial probe by triple staining. probes and cell membrane monomer cyanine nucleic acid stain TO-PROTM-1 were used to observe the protective effect of different concentrations of the atomized liquid composition on CSE-induced BEAS-2B cell membrane and mitochondrial damage. Hoechst33342 can bind to the nucleus of living cells to produce uniform light blue fluorescence. Mitochondrial probe The probes can mark active mitochondria in cells, reflecting the mitochondrial membrane potential. The stronger the mitochondrial activity, the higher the membrane potential, and the stronger the fluorescence intensity. After the cells are damaged and dead, the cell membrane permeability increases, and it combines with the dye YO-PROTM-1 to produce bright green fluorescence, marking the damaged and dead cells.

[0253] 3. Protective effect of the nebulized liquid composition on CSE-induced ROS damage in BEAS-2B cells

[0254] The cells were inoculated in a black transparent bottom 96-well plate. Five groups were set up in the experiment, including a blank group, a CSE intervention group, a 0.01% nebulized liquid composition, a 0.05% nebulized liquid composition, and a 0.1% nebulized liquid combination group. The experiment was repeated 3 times. Fresh culture medium was added to the blank control group, 5% CSE was added to the CSE intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 5% CSE were added to the nebulized liquid combination group for co-culture for 24 hours. Using the BioTek Cytaion5 high-content imager, the protective effect of different concentrations of nebulized liquid compositions on CSE-induced ROS damage in BEAS-2B was preliminarily evaluated at the cellular level by double staining with Hoechst33342 and oxidative stress marker CellROXOxidativeStressReagents. CellROXGreenRergent is a fluorescent probe for detecting reactive oxygen species (ROS) in living cells. It has no fluorescence or very weak fluorescence in the reduced state. When cells are damaged, a large amount of reactive oxygen species is produced, which can oxidize the CellROX dye and bind to DNA to produce bright green fluorescence. The signal is localized in the cell nucleus and mitochondria.

[0255] 4. Effect of nebulizer composition on CSE-induced BEAS-2B inflammation

[0256] The cells were inoculated in 6-well plates. Five groups were set up in the experiment, including blank group, CSE intervention group, 0.01% nebulized liquid composition, 0.05% nebulized liquid composition, and 0.1% nebulized liquid composition. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, 5% CSE was added to the CSE intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 5% CSE were added to the nebulized liquid composition group for co-culture for 24 hours. The cells were collected and IL-1β, IL-6, and TNF-α in the cells were detected by enzyme-linked immunosorbent assay.

[0257] 5. CCK-8 method was used to detect the effect of different concentrations of TNF-α on the survival rate of BEAS-2B cells

[0258] Cells were inoculated in 96-well plates, and 8 groups were set up in the experiment, including zero-adjustment group, blank group, 5, 10, 20, 30, 40, and 50 μg / L TNF-α groups. Each group was set up with 5 wells, and the experiment was repeated 3 times. The zero-adjustment group did not inoculate cells but only added fresh culture medium, the blank group added fresh culture medium, and the other groups added different concentrations of TNF-α for 24 hours. The cell supernatant was discarded, washed once with 100 μL / well PBS, and 100 μL / well BEGM culture medium containing 10% CCK-8 reagent was added. After incubation in a cell culture incubator at 37°C and 5% CO2 for 1 hour, CCK-8 was measured at a wavelength of 450nm on an ELISA reader to calculate the OD value.

[0259] Calculation of cell survival rate: survival rate = (OD value of experimental well - OD value of control well) / (OD value of control well - OD value of blank well) * 100%.

[0260] 6. Protective effect of the nebulized liquid composition on TNF-α-induced BEAS-2B cell membrane and mitochondrial damage

[0261] The cells were seeded in a black transparent bottom 96-well plate. Five groups were set up in the experiment, namely, blank group, TNF-α intervention group, 0.01% nebulized liquid composition group, 0.5% nebulized liquid composition group, and 1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank control group, 20μg / L TNF-α was added to the TNF-α intervention group, and 0.01%, 0.05%, and 0.1% nebulized liquid composition and 20μg / L TNF-α were added to the nebulized liquid composition group for co-culture for 24 hours. The BioTek Cytaion5 high-content imager was used to simultaneously stain the cell nucleus with Hoechst33342 and the mitochondrial probe by triple staining. Probes and cell membrane monomeric cyanine nucleic acid stains -1, observe the protective effect of different concentrations of nebulized liquid compositions on BEAS-2B cell membrane and mitochondrial damage induced by TNF-α.

[0262] 7. Protective effect of the nebulized liquid composition on TNF-α-induced ROS in BEAS-2B cells

[0263] The cells were seeded in a black transparent bottom 96-well plate. Five groups were set up in the experiment, namely, blank group, TNF-α intervention group, 0.01% nebulized liquid composition group, 0.05% nebulized liquid composition group, and 1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank control group, 20μg / L TNF-α was added to the TNF-α intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 20μg / L TNF-α were added to the nebulized liquid composition group for co-culture for 24h. The protective effect of different concentrations of nebulized liquid composition on CSE-induced ROS damage of BEAS-2B cells was preliminarily evaluated at the cellular level by double staining of Hoechst33342 and oxidative stress marker CellROXOxidativeStressReagents using BioTekCytaion5 high-content imager.

[0264] 8. Effect of nebulized liquid composition on TNF-α-induced BEAS-2B inflammation

[0265] The cells were inoculated in 6-well plates. Five groups were set up in the experiment, including blank group, CSE intervention group, 0.01% nebulized liquid composition group, 0.5% nebulized liquid composition group, and 1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, 20 μg / L TNF-α was added to the TNF-α intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 20 μg / L TNF-α were added to the nebulized liquid composition group for co-culture for 24 hours. The cells were collected and IL-1β and IL-6 in the cells were detected by enzyme-linked immunosorbent assay.

[0266] 9. Evaluating the effect of different concentrations of CSE on the survival rate of mouse alveolar macrophages (MH-S) by CCK-8 method

[0267] Cells were inoculated in 96-well plates, and 8 groups were set up in the experiment, including zero-adjustment group, blank group, 0.5%, 1%, 2%, 5%, 10%, and 20% CSE groups. Each group was set up with 5 wells, and the experiment was repeated 3 times. The zero-adjustment group did not inoculate cells but only added fresh culture medium, the blank group added fresh culture medium, and the other groups added different concentrations of CSE for 24 hours. The cell supernatant was discarded, washed once with 100 μL / well PBS, and 100 μL / well 1640 culture medium containing 10% CCK-8 reagent was added. After incubation in a cell culture incubator at 37°C and 5% CO2 for 1 hour, CCK-8 was measured at a wavelength of 450nm on an ELISA reader, and the OD value was calculated.

[0268] Calculation of cell survival rate: survival rate = (OD value of experimental well - OD value of control well) / (OD value of control well - OD value of blank well) * 100%.

[0269] 10. Protective effect of the nebulized liquid composition on CSE-induced MH-S cell membrane and mitochondrial damage

[0270] The cells were inoculated in a black transparent bottom 96-well plate. The experiment was set up with 5 groups, including a blank group, a CSE intervention group, a 0.01% nebulized liquid composition group, a 0.05% nebulized liquid composition group, and a 0.1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, 5% CSE was added to the CSE intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 5% CSE were added to the nebulized liquid composition group for co-culture for 24 hours. The BioTek Cytaion5 high-content imager was used to simultaneously stain Hoechst33342 and mitochondrial probes by triple staining. Probes and cell membrane monomeric cyanine nucleic acid stains -1 Protective effect of different concentrations of nebulized liquid composition on CSE-induced MH-S cell membrane and mitochondrial damage.

[0271] 11. Protective effect of the aerosol liquid composition on CSE-induced ROS in MH-S cells

[0272] The cells were seeded in a black transparent bottom 96-well plate. Five groups were set up in the experiment, namely, blank group, CSE intervention group, 0.01% nebulized liquid composition group, 0.05% nebulized liquid composition group, and 0.1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, 5% CSE was added to the CSE intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 5% CSE were added to the nebulized liquid composition group for co-culture for 24 hours. The BioTekCytaion5 high-content imager was used to preliminarily evaluate the protective effect of different concentrations of nebulized liquid composition on CSE-induced ROS damage of MH-S cells at the cellular level by double staining Hoechst33342 and oxidative stress marker ROS.

[0273] 12. Effect of nebulizer composition on CSE-induced MH-S inflammation

[0274] The cells were inoculated in 6-well plates, and the experiment was set up in 5 groups, including blank group, CSE intervention group, 0.01% nebulized liquid composition group, 0.05% nebulized liquid composition group, and 0.1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, 5% CSE was added to the CSE intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 5% CSE were added to the nebulized liquid composition group for co-culture for 24 hours. The cells were collected, and IL-1β and TNF-α in the cell suspension were detected by enzyme-linked immunosorbent assay.

[0275] 13. CCK-8 method was used to detect the effect of different concentrations of TNF-α on the survival rate of MH-S cells

[0276] Cells were inoculated in 96-well plates, and 8 groups were set up in the experiment, including zero-adjustment group, blank group, 5, 10, 20, 30, 40, and 50 μg / L TNF-α groups. Each group was set up with 5 wells, and the experiment was repeated 3 times. The zero-adjustment group did not inoculate cells but only added fresh culture medium, the blank group added fresh culture medium, and the other groups added different concentrations of TNF-α for 24 hours. The cell supernatant was aspirated, washed once with 100 μL / well PBS, and 100 μL / well 1640 culture medium containing 10% CCK-8 reagent was added. After incubation in a cell culture incubator at 37°C and 5% CO2 for 1 hour, CCK-8 was measured at a wavelength of 450nm on an ELISA reader to calculate the OD value.

[0277] Calculation of cell survival rate: survival rate = (OD value of experimental well - OD value of control well) / (OD value of control well - OD value of blank well) * 100%.

[0278] 14. Protection of the aerosol liquid composition against TNF-α-induced MH-S cell membrane and mitochondrial damage

[0279] The cells were inoculated in a black transparent bottom 96-well plate. The experiment was set up with 5 groups, including a blank group, a TNF-α intervention group, a 0.01% nebulized liquid composition group, a 0.05% nebulized liquid composition group, and a 0.1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, 20 μg / L TNF-α was added to the TNF-α intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 20 μg / L TNF-α were added to the nebulized liquid composition group for co-culture for 24 hours. The BioTek Cytaion5 high-content imager was used to simultaneously stain Hoechst33342 and mitochondrial probe by triple staining. Probes and cell membrane monomeric cyanine nucleic acid stains -1 Protective effect of different concentrations of nebulized liquid composition on TNF-α-induced MH-S cell membrane and mitochondrial damage.

[0280] 15. Effect of the nebulized liquid composition on TNF-α-induced ROS in MH-S cells

[0281] The cells were seeded in a black transparent bottom 96-well plate. Five groups were set up in the experiment, namely, blank group, TNF-α intervention group, 0.01% nebulized liquid composition group, 0.05% nebulized liquid composition group, and 0.1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, 20 μg / L TNF-α was added to the TNF-α intervention group, and 0.01%, 0.05%, 0.1% nebulized liquid composition and 20 μg / L TNF-α were added to the nebulized liquid composition group for co-culture for 24 h. The protective effect of different concentrations of nebulized liquid composition on TNF-α-induced ROS damage of MH-S cells was preliminarily evaluated at the cellular level by double staining of Hoechst33342 and oxidative stress marker ROS using BioTekCytaion5 high-content imager.

[0282] 16. Effects of the aerosol liquid composition on MDA and SOD in H2O2-induced oxidative stress in MH-S cells

[0283] The cells were inoculated in 6-well plates, and the experiment was set up in 5 groups, including blank group, H2O2 intervention group, 0.01% nebulized liquid composition group, 0.05% nebulized liquid composition group, and 0.1% nebulized liquid composition group. The experiment was repeated 3 times. Fresh culture medium was added to the blank group, fresh culture medium was added to the blank group, 100 μmol / L was added to the H2O2 intervention group for 4 hours, and 0.01%, 0.05%, and 0.1% nebulized liquid composition were added to the nebulized liquid composition group for 24 hours, and 100 μmol / L was added for 4 hours. The cells were collected and tested according to the instructions of the MDA and SOD enzyme-linked immunosorbent assay kits.

[0284] 3. Experimental Results

[0285] 1. Mouse cough suppression experiment

[0286] Compared with the model group, the cough frequency of the high-dose group of the nebulized liquid composition was significantly reduced (P<0.05), and the low-dose group and the medium-dose group had a certain trend of reduction; the cough latency of the medium-dose group and the high-dose group of the nebulized liquid composition had a tendency to be prolonged, but there was no statistically significant difference (P>0.05). The results are shown in Table 8.

[0287] Table 8 Atomized liquid composition atomized inhalation cough test results ( n=12)

[0288]

[0289] Note: 1. Compared with the model group, *P<0.05, **P<0.01.

[0290] 2. The experiment used the animal inhalation of the aerosol liquid composition as the animal's inhalation dose, and the calculation formula was: inhalation dose (mg / kg) = aerosol concentration (mg / Lair) × breathing volume per minute (L / min) × aerosol time (min) / body weight (kg). The monitor detected that the aerosol concentration of the aerosol liquid composition was 7.62g / m 3 , BW: average body weight of mice = 0.020 kg; theoretical minute respiratory volume (RMV) of mice: = 0.608 × 0.020 × 0.852 = 0.022 L / min, dosage: the dosage of the nebulized aerosol liquid composition for 5 minutes is 41.91 mg / kg; the dosage of the nebulized aerosol liquid composition for 10 minutes is 83.82 mg / kg; the dosage of the nebulized aerosol liquid composition for 20 minutes is 167.64 mg / kg.

[0291] 2. Protective effect of the nebulized liquid composition on CSE-induced BEAS-2B cell membrane and mitochondrial damage

[0292] Hoechst staining results showed that CSE caused cell damage, the number of cells decreased, and the nucleus showed irregular changes such as shrinkage and deformation. MitoTracker mitochondrial staining showed that the fluorescence intensity was weakened and irregular changes occurred. The YO-PRO fluorescence results showed that the CSE group had cell membrane damage. Compared with the CSE group, the number of cells increased with the increase of the concentration of the atomized liquid composition, and the mitochondrial fluorescence intensity had an increasing trend. Results are shown in Figure 7 . Figure 7 The results showed that the nebulized liquid composition has a protective effect on CSE-induced BEAS-2B cell membrane and mitochondrial damage.

[0293] 3. Protective effect of the aerosol liquid composition on CSE-induced ROS damage in BEAS-2B

[0294] Compared with the blank group, bright green fluorescence was observed in the CSE group, which overlapped with the blue signal of the cell nucleus, indicating that CSE could induce BEAS-2B cells to produce ROS. Compared with the CSE group, the fluorescence of the nebulized liquid composition decreased with the increase of the administration concentration, especially the fluorescence of the 0.1% nebulized liquid composition group decreased significantly. Figure 8 . Figure 8 The nebulized liquid composition showed the protective effect against ROS damage caused by CSE in BEAS-2B

[0295] 4. Effect of nebulizer composition on CSE-induced BEAS-2B inflammation

[0296] Compared with the blank group, the IL-1β and TNF-α levels in the CSE group were significantly increased (P<0.01, P<0.001). The IL-1β level in the 0.1% nebulized liquid composition group was significantly reduced (P<0.05), and the 0.01% nebulized liquid composition group and the 0.05% nebulized liquid composition group had a certain downward trend, and TNF-α had a certain downward trend. In this experiment, the IL-6 level in the CSE group showed an upward trend, but no significant difference was found (P>0.05). See Table 9.

[0297] Table 9 Effects of nebulized liquid composition on IL-1β and TNF-α of BEAS-2B cells induced by CSE ( n=3)

[0298]

[0299] Note: Compared with the blank group, **P<0.01, ***P<0.001;

[0300] 5. CCK-8 method was used to detect the effect of different concentrations of TNF-α on the survival rate of BEAS-2B cells

[0301] As the concentration of TNF-α increased, the cell survival rate gradually decreased. In the experiment, 20 μg / L TNF-α was selected as the modeling concentration for subsequent experiments, which can both reasonably stimulate the concentration and ensure the cell survival rate as much as possible. The results are shown in Table 10.

[0302] Table 10 Effect of TNF-α on BEAS-2B cell survival rate ( n=3)

[0303]

[0304] Note: Compared with the blank group, * P<0.05, *** P<0.001.

[0305] 6. Protective effect of the atomized liquid composition on TNF-α-induced BEAS-2B cell membrane and mitochondrial damage

[0306] Compared with the blank group, the TNF-α group showed less nuclear shrinkage of BEAS-2B cells, decreased orange fluorescence signal of mitochondria, and increased green fluorescence signal of cell membrane, indicating that TNF-α caused decreased mitochondrial membrane potential, increased cell membrane permeability, and cell damage in BEAS-2B cells. Compared with the TNF-α group, the orange fluorescence signal tended to increase with the increase of the concentration of the nebulized liquid composition, while the green fluorescence signal of the cell membrane tended to decrease. Fig. 9 . Fig. 9 The protective effect of the nebulized liquid composition on TNF-α-induced BEAS-2B cell membrane and mitochondrial damage

[0307] 7. Protective effect of the nebulized liquid composition on TNF-α-induced ROS damage in BEAS-2B cells

[0308] Compared with the blank group, the green fluorescence signal of the TNF-α group increased significantly. The Merge diagram showed that the green fluorescence overlapped with the blue fluorescence of Hoechst, suggesting that TNF-α can induce an increase in the content of reactive oxygen species (ROS) in BEAS-2B cells. Compared with the TNF-α group, the atomized liquid compositions of different concentrations reduced the content of intracellular ROS to a certain extent. The results are shown in Fig.10 . Fig.10 The nebulized liquid composition showed a protective effect against ROS damage caused by TNF-α in BEAS-2B

[0309] 8. Effect of nebulized liquid composition on TNF-α-induced BEAS-2B inflammation

[0310] Compared with the blank group, the inflammatory factor IL-1β content in the TNF-α stimulation group increased significantly (P<0.01), and the IL-1β content in the 0.05% atomized liquid composition group decreased significantly (P<0.05), and the 0.1% group showed a significant downward trend; the inflammatory factor IL-6 content in the TNF-α stimulation group only showed an upward trend, and no statistical difference was found. Different concentrations of atomized liquid compositions had no significant effect on IL-6 content. The results are shown in Table 11.

[0311] Table 11 Effect of the atomized liquid composition on TNF-α-induced BEAS-2B inflammatory factors ( n=3)

[0312]

[0313] Note: Compared with the blank group, ** P<0.01; compared with TNF-α group, # P<0.05.

[0314] 9. Evaluating the effect of different concentrations of CSE on the survival rate of mouse alveolar macrophages (MH-S) by CCK-8 method

[0315] Compared with the blank group, CSE intervention for 24 hours had a certain effect on the number of MH-S cells. With the increase of CSE concentration, the damage of CSE to MH-S cells was greater and the cell survival rate decreased. When the CSE concentration was 10%, the cell survival rate decreased significantly (P<0.05). The effect of 5% CSE on the survival rate of MH-S cells was close to 50%. In order to better carry out subsequent experimental research and ensure cell survival rate, 5% was selected as the concentration of CSE damage experiment. The results are shown in Table 12.

[0316] Table 12 Effects of different concentrations of CSE on MH-S cell activity ( n=3)

[0317]

[0318] Note: Compared with the blank group * P<0.05, *** P<0.001.

[0319] 10. Protective effect of the atomized liquid composition on MH-S cell membrane and mitochondrial damage induced by CSE liquid

[0320] Compared with the blank group, the number of cells in the CSE group decreased, the nucleus shrank and deformed, the intensity of mitochondrial orange fluorescence weakened, and a large amount of scattered green fluorescence was visible, indicating that CSE induced mitochondrial damage in MH-S cells, enhanced cell membrane permeability, and cell damage. The high-dose nebulized liquid composition can improve the cell membrane and mitochondrial damage induced by CSE to a certain extent. Results are shown in Fig.11 . Fig.11 The results show that different concentrations of nebulized liquid compositions have protective effects on CSE-induced cell membrane and mitochondrial damage.

[0321] 11. Protective effect of the aerosol liquid composition on ROS damage caused by CSE in MH-S

[0322] Compared with the blank group, the green fluorescence in the CSE group increased significantly, and the Merge results showed that the green fluorescence overlapped with the cell nucleus, suggesting that CSE can stimulate MH-S cells to produce a large amount of reactive oxygen species (ROS), causing MH-S cell damage. After administering different concentrations of atomized herbal liquid, the amount of ROS in the cells decreased to a certain extent. Fig.12 . Fig.12 Show the protective effect of different concentrations of nebulized liquid compositions on CSE-induced ROS damage 12. Effect of nebulized liquid compositions on CSE-induced inflammatory factors in MH-S cells

[0323] Compared with the blank group, the IL-1β content in the high-dose group of the atomized liquid composition was significantly reduced, and the TNF-α content showed a decreasing trend, but no statistical difference was found. The results are shown in Table 13.

[0324] Table 13 Effects of nebulized liquid composition on CSE-induced MH-S cell inflammatory factors ( n=3)

[0325]

[0326] Note: Compared with the blank group ** P<0.01; compared with CSE group # P<0.05.

[0327] 13. CCK-8 method was used to detect the effect of different concentrations of TNF-α on the survival rate of MH-S cells

[0328] As the concentration of TNF-α increased, the survival rate of MH-S cells gradually decreased. In order to select a reasonable stimulation concentration while ensuring the cell survival rate as much as possible, 20 μg / L TNF-α was selected as the modeling concentration for subsequent experiments. The results are shown in Table 14.

[0329] Table 14 Effect of TNF-α on the survival rate of MH-S cells ( n=3)

[0330]

[0331] Note: Compared with the blank group * P<0.05, ** P<0.01.

[0332] 14. Protection of the aerosol liquid composition against TNF-α-induced MH-S cell membrane and mitochondrial damage

[0333] Compared with the blank group, the number of cells in the TNF-α group decreased, the mitochondrial fluorescence intensity decreased, and the green fluorescence of the cell membrane increased, indicating that TNF-α can induce damage to the mitochondria and cell membrane of MH-S cells. The high-concentration atomized liquid composition has a certain improvement on mitochondria, but has no obvious effect on cell membrane damage. Fig.13 . Fig.13 The results show that different concentrations of nebulized liquid compositions have protective effects on TNF-α-induced MH-S cell membrane and mitochondrial damage.

[0334] 15. Protective effect of the aerosol liquid composition on TNF-α-induced ROS damage in MH-S cells

[0335] Compared with the blank group, the TNF-α group showed scattered ROS green fluorescence, indicating that TNF-α can induce MH-S cells to produce reactive oxygen species, which has a certain damaging effect on MH-S. The atomized liquid composition of different concentrations has a certain inhibitory effect on the production of ROS. The results are shown in Fig.14 . Fig.14 Showing the protective effect of different concentrations of nebulized liquid compositions on TNF-α-induced ROS damage in MH-S cells

[0336] 16. Effects of the aerosol liquid composition on MDA and SOD in MH-S cells induced by H2O2

[0337] Excessive ROS produced in cells causes lipid peroxidation, produces a large amount of MDA products, and consumes intracellular antioxidant enzymes such as SOD, resulting in reduced antioxidant enzyme activity. Compared with the blank group, the MDA content in the H2O2 group was significantly increased (P<0.01), and the SOD activity was significantly reduced (P<0.01); compared with the H2O2 group, the MDA level of the high-dose nebulized liquid composition cells was significantly reduced (P<0.05), and the SOD activity of different nebulized liquid composition groups showed an increasing trend, but no statistical difference was found. The results are shown in Table 15.

[0338] Table 15 Effect of aerosol liquid composition on H2O2-induced MDA and SOD in MH-S cells ( n=3)

[0339]

[0340] Note: Compared with the blank group ** P<0.01; compared with H2O2 group # P<0.05.

[0341] IV. Conclusion

[0342] 1. The results of the mouse cough-suppressing experiment showed that compared with the model group, the cough latency in the positive drug group was significantly prolonged (P>0.05 or P<0.01), and the number of coughs was significantly reduced (P<0.01); the number of coughs in the high-dose group of the nebulized liquid composition was significantly reduced (P<0.01 or P<0.05). This shows that the nebulized liquid composition has a significant inhibitory effect on ammonia-induced cough and reduces the number of coughs, suggesting that the nebulizer device and the nebulized liquid composition have a certain antitussive effect.

[0343] 2. The BEAS-2B cell injury and inflammation models induced by exogenous CSE and endogenous TNF-α were established respectively. The nebulized liquid composition had a certain improvement effect on CSE-induced BEAS-2B cell injury and reduced the content of IL-1β. The nebulized liquid composition can improve CSE- and TNF-α-induced BEAS-2B cell injury and inflammation.

[0344] 3. Establish an exogenous CSE-induced MH-S cell injury and inflammation model. The nebulized liquid composition has a certain improvement effect on CSE-induced MH-S cell injury and can reduce the content of CSE-induced IL-1β. The nebulized liquid composition can improve CSE-induced MH-S cell injury and inflammation.

[0345] 4. Establishing the H2O2-induced MH-S cell oxidative stress model, the atomized liquid composition can inhibit the production of H2O2-induced MH-S cell reactive oxygen species, reduce the MDA content, and promote SOD activity. The atomized liquid composition has the effect of inhibiting H2O2-induced MH-S cell oxidative stress.

[0346] 5. The atomized liquid composition can be used to improve respiratory inflammation caused by smoking.

[0347] Experimental Example 7: Pharmacokinetics and tissue distribution experiment of atomization device and atomization liquid composition

[0348] A large amount of propylene glycol is added to the atomized liquid composition. Propylene glycol has low toxicity. As an external preparation, propylene glycol may irritate and sensitize the skin. Therefore, the pharmacokinetics and tissue distribution of the atomized liquid composition after atomization inhalation are studied.

[0349] 1. Preparation of atomized liquid composition

[0350] According to the formula and method of Example 1, atomized liquid composition 1 was prepared.

[0351] 2. Experimental Methods

[0352] 1. Study on atomized particle size of atomized liquid composition

[0353] The atomizing suction machine is connected to the atomizing compressor and the atomizing device of the present invention to ensure that the atomizing suction machine does not separate from the atomizing compressor under the specific gas flow rate and pressure of the compressor, and is connected to the inlet of the laser diffractometer, and the outlet is connected to the external filter, and the external filter is connected to the vacuum pump. Connect the flow meter at the inlet, turn on the vacuum pump, and adjust the gas flow rate at the inlet to stabilize at 15L / min (±5%). After adjusting the gas flow rate, open the software (Spraytec softwareversion 3.20, Malvern) on the computer and set the SOP (test mode: continuous mode; refractive index of particles: 1.33; dispersion medium: air; refractive index: 1.00), and then start testing the light path background. After the background test is qualified, the smoke outlet pipe of the atomizing suction machine is tightly connected to the inlet end of the induction port. Set the parameters, RTE-CY02 atomizing suction machine: suction pressure 1.5kPa, 5s per suction, suction interval 5s, and dual channels are opened at the same time. Oral and nasal exposure tower: ventilation volume 0.5L / unit. Malvern Panalytical Spraytec Spray Particle Size Analyzer: air compressor flow rate 15L / min, sampling time 5min. Then turn on the atomizer to atomize and test the particle size of the drug particles. After testing for 5min, turn off the atomizer. During the analysis, continuously monitor the particle size distribution (Dv10, Dv 50, Dv 90).

[0354] 2. Mucosal irritation test of aerosol liquid composition in rats by aerosol inhalation

[0355] The animals were fed adaptively for 5 days, and the weight of the animals was weighed on the day of grouping. Females and males were grouped separately. The number of animals involved in the grouping: 14 female mice; 14 male mice, randomly divided into blank control group and nebulized inhalation administration group according to the weight of the animals (grouping is shown in Table 1). When administering, the nebulizer suction machine was connected to the oral and nasal exposure device, and the oral and nasal exposure device was set: the ventilation volume of the DSI oral and nasal exposure tower was 0.5L / mouse, the suction pressure of the RTE-CY02 nebulizer suction machine was 1.5kPa, each suction was 5s, the suction interval was 5s, and the two channels were opened at the same time. The animals were fixed in the oral and nasal exposure tower, and the drug was administered continuously for 60min, once a day, for 7 consecutive days. The blank control group was given air in the same way. 24h after the last administration, 8 animals (half male and half female) were taken from each group, and the remaining animals were treated after 7 days of drug withdrawal.

[0356] The following indicators were tested during the experiment:

[0357] 1) Gross observation: Before each administration, observe the rats’ nasal and oral mucosa for redness, swelling, bleeding, ulcers, etc. within the visible range; immediately after administration, observe the animals’ local irritation symptoms (such as asthma, cough, vomiting, suffocation, etc.) as well as their general state, behavior, and physical signs.

[0358] 2) White blood cell count and classification of bronchoalveolar lavage fluid: 24 hours after the last administration, 8 animals (half male and half female) were selected from each group, the chest cavity was exposed, the right lobe was ligated, and the left lung was lavaged with normal saline. The solution was slowly injected, allowed to rest for 30 seconds, and then slowly withdrawn. 1 mL / time, lavage was performed twice, and the lavage speed was 2 mL / min (30 seconds / time). The lavage fluid was combined and centrifuged (1000 rpm, 10 minutes). The precipitate was resuspended with 0.5 mL D-Hanks buffer solution, and the white blood cell count and classification were determined. After the recovery period of the remaining animals, the white blood cell count and classification were determined in the same way.

[0359] 3) Histopathological examination: After the animals were killed, the local mucosal tissues of the respiratory tract were observed for congestion, edema, ulcers, etc. The nasal mucosa, oral mucosa, pharynx, trachea and lungs of the animals were fixed in 10% formalin solution. The nasal cavity was decalcified with 5% formic acid, and all tissues were dehydrated, paraffin-embedded, sliced, and finally stained with hematoxylin-eosin (HE).

[0360] 3. Active systemic allergy test on guinea pigs by aerosol inhalation of aerosol liquid composition

[0361] The animals were grouped before administration, and the group weights were weighed on the day of grouping. Females and males were grouped separately. Number of animals involved in grouping: female mice: 12; male mice: 12. (Group weights are shown in Table 16)

[0362] Table 16 Group weight is as follows ( n=4)

[0363]

[0364] Sensitization stage: RTE-CY02 nebulizer suction machine: suction pressure 1.5kPa, 5s per suction, 5s interval between suctions, dual channels open at the same time. Oral and nasal exposure tower: ventilation volume 1L / animal. Animals were fixed in the oral and nasal exposure system, and the nebulizer liquid composition was nebulized for 30min, once every other day, for a total of 3 times.

[0365] Provocation phase: RTE-CY02 nebulizer: suction pressure 1.5 kPa, 5 seconds per suction, 5 seconds between suctions, dual channels open simultaneously. Oral and nasal exposure tower: ventilation volume 1 L / animal. Animals were fixed in the oral and nasal exposure system, and the nebulizer liquid composition was nebulized for 60 minutes on the 14th and 21st days after the last sensitization.

[0366] The negative control group was administered by inhalation through the mouth and nose after nebulized air. Sensitization: 30 minutes of nebulized air administration, once every other day, for a total of 3 times. Challenge: 60 minutes of nebulized air administration on the 14th and 21st days after the last sensitization. The positive control group was administered by inhalation through the mouth and nose after nebulized ovalbumin solution. Sensitization: Take ovalbumin, prepare it into 2mg / mL sensitization solution with sodium chloride injection, nebulize the sensitization solution for 15 minutes, and administer it once every other day, for a total of 3 times. Challenge: Take ovalbumin, prepare it into 4mg / mL stimulating solution with sodium chloride injection, and stimulate it with nebulized solution for 30 minutes on the 14th and 21st days after the last sensitization.

[0367] The following indicators were observed during the experiment: Animal weight and general symptoms observation: The weight of each animal in each group was weighed and recorded on each sensitization and challenge day. Data are expressed as mean ± standard deviation ( X ± SD), SPSS20.0 was used for statistical analysis, and T-test was used for comparison between groups. The statistical significance standard for statistical tests was P < 0.05. During the sensitization period, the general symptoms of the animals were observed and recorded daily. Systemic allergic reaction: Immediately to 30 minutes after stimulation, observe whether the animals have the symptoms shown in Table 17. Record and calculate the incidence of allergic reactions.

[0368] Table 17 Allergic reaction symptoms

[0369]

[0370]

[0371] 4. Metabolic kinetics and tissue distribution of aerosol liquid composition

[0372] 4.1 Preliminary determination of chemical component concentrations in the atomized liquid composition

[0373] Blank serum: Rats were given normal drinking water and fasted for 24 hours. The animals were anesthetized with 10% chloral hydrate, and blood was collected from the abdominal aorta and collected into EDTA anticoagulant tubes. The blood was centrifuged at 3500rpm for 10 minutes at 4°C. The plasma was mixed and transferred into 1.5mL EP tubes and stored at -80°C for testing. The lungs were weighed and respectively transferred into 2mL EP tubes and stored at -80°C for testing.

[0374] Single dose: fast overnight the day before administration, drink water normally. 5min, 15min, 30min, 45min, 1h, 2h, 4h, 6h after inhalation administration, blood was collected through the eye socket, about 200μL each time, and collected into EDTA anticoagulation EP tubes, centrifuged at 3500rpm for 10min at 4℃, and the plasma was transferred to 1.5mL EP tubes, stored at -80℃ for testing; (after weighing the lungs, they were placed in 2mL EP tubes, stored at -80℃ for testing).

[0375] Multiple administration: animals were given normal drinking water and feed, and continuous aerosol administration for 7 days. The animals were fasted overnight and given normal drinking water the day before the last administration. Blood was collected from the eye sockets at 5min, 15min, 30min, 45min, 1h, 2h, 4h, 6h, and 24h after the last administration. About 200μL of blood was collected from each animal each time and placed in EDTA anticoagulated EP tubes. The blood was centrifuged at 3500rpm for 10min at 4℃, and the plasma was transferred to 1.5mL EP tubes and stored at -80℃ for testing. The lung tissues of male and female rats that were given multiple administrations were taken, weighed, and placed in 2mL EP tubes, stored at -80℃ for testing.

[0376] Animal tissue processing: After the rats are anesthetized, the blood stains are wiped clean, and then the complete lung tissue is taken. The removed lung tissue is rinsed with running saline, the water is absorbed with filter paper, weighed, and stored at -80°C. When testing, each tissue block is frozen and ground, and 1mL of nimodipine-containing extract is added respectively. After vortex mixing, ultrasonic extraction is performed, and 800μL of supernatant is taken after centrifugation at 13000rpm for 10min. Add another 500μL of extract, vortex, ultrasonic, and centrifuge at 13000rpm for 10min, and then take 400μL of supernatant. Mix the two supernatants, spin dry, add 100μL of reconstitution solution, vortex, centrifuge, and take the supernatant for sampling and analysis.

[0377] 6.2 Pharmacokinetics and tissue distribution studies in rats after administration of the nebulized liquid composition

[0378] Dosage and grouping: The pharmacokinetic study in rats is divided into single administration and continuous administration. One dose group is set up, namely the 60-min aerosol administration group. Both the single-dose group and the continuous administration group have 6 animals, half of which are male and half are female. The continuous administration group is aerosolized once a day, 60 minutes each time, and administered 7 times in succession. After the SD rats were purchased, they were adapted to the rat room of the animal center for at least 5 days, with a temperature of (23±1)℃, a humidity of 50%±15%, and a 12-hour day and night alternation. The rats were fasted overnight the day before administration and drank water normally.

[0379] Single-dose pharmacokinetics: Oral and nasal exposure system nebulized inhalation administration, administration time 1 hour, to determine the pharmacokinetic characteristics of the test component in rats after administration. The experiment began after the rats were adaptively fed. Blood was collected before administration, 5, 10, 20, 40min, 1, 2, 4, 6, 8, 10, 12 and 24h after administration, and the blood was placed in EDTA anticoagulated EP tubes, and the plasma was separated and stored at -80℃ until analysis. After analysis and determination, the pharmacokinetic parameters such as T1 / 2, Cmax, Tmax, AUC, etc. of the test component were calculated by pharmacokinetic software.

[0380] Continuous administration pharmacokinetics: Pharmacokinetic parameters of the test component after the first and last administration, and analysis of whether the test component has an accumulation effect in rats. Six rats were used for the experiment after adaptive feeding, and the drug was administered for 7 consecutive days. Blood was collected before the first administration, 5, 10, 20, 40 minutes, 1, 2, 4, 6, 8, 10, 12 and 24 hours after administration; before the last administration, 10, 20, 40 minutes, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72 hours after administration; and 24 hours after each administration in between, and the blood was placed in an EDTA anticoagulated EP tube, and the plasma was separated and stored at -80°C until analysis. After analysis and determination, the pharmacokinetic parameters such as T1 / 2, Cmax, Tmax, AUC, etc. after the first and last administration of the test component were calculated by pharmacokinetic software, and a two-sample equal variance t-test analysis was performed.

[0381] Tissue distribution and residue study: used to investigate the distribution of the test component in rat tissues and organs after a single dose, as well as its elimination in tissues and organs. 54 SD rats, half male and half female, were used for the experiment after adaptive feeding. Before administration (i.e., blank rats), 20min, 40min, 60min, 4h, 8h, 24h, 7d, and 14d after a single dose, 6 animals were treated for each treatment, and blood, as well as tissues and organs such as the throat, trachea, and lungs were collected and stored at -80°C until analysis.

[0382] 3. Experimental Results

[0383] 1. Study on atomized particle size of atomized liquid composition

[0384] The atomized liquid composition atomized particle size distribution results are as follows: Dv (10) = 0.4 μm, Dv (50) = 0.5337 μm, Dv (90) = 345 μm, transmittance = 14.6%, particle size distribution diagram is shown in Fig.15 . Fig.15 It is the aerosol particle size distribution diagram of the atomized liquid composition.

[0385] Table 18 Atomized liquid composition atomized particle size distribution

[0386] <![CDATA[Particle size (μ m )]]> Cumulative volume % Volume % Particle size (μm) Cumulative volume % Volume % Particle size (μm) Cumulative volume % Volume % 0.117 0.00 0.00 2.51 74.55 0.00 54.12 74.55 0.00 0.136 0.00 0.00 2.93 74.55 0.00 63.10 74.55 0.00 0.158 0.00 0.00 3.41 74.55 0.00 73.56 74.55 0.00 0.185 0.00 0.00 3.98 74.55 0.00 85.77 74.55 0.00 0.215 0.00 0.00 4.64 74.55 0.00 100.00 74.55 0.00 0.251 0.02 0.02 5.41 74.55 0.00 116.59 74.55 0.00 0.293 0.25 0.23 6.31 74.55 0.00 135.94 74.55 0.00 0.341 2.01 1.76 7.36 74.55 0.00 158.49 74.56 0.02 0.398 9.50 7.50 8.58 74.55 0.00 184.79 74.76 0.20 0.464 27.74 18.23 10.00 74.55 0.00 215.44 75.73 0.97 0.541 52.14 24.40 11.66 74.55 0.00 251.19 78.46 2.73 0.631 68.82 16.68 13.59 74.55 0.00 292.87 83.42 4.96 0.736 73.95 5.13 15.85 74.55 0.00 341.46 89.59 6.17 0.858 74.53 0.58 18.48 74.55 0.00 398.11 94.99 5.40 1.00 74.55 0.02 21.54 74.55 0.00 464.16 98.31 3.32 1.17 74.55 0.00 25.12 74.55 0.00 541.17 99.67 1.36 1.36 74.55 0.00 29.29 74.55 0.00 630.96 99.98 0.31 1.58 74.55 0.00 34.15 74.55 0.00 735.64 100.00 0.02 1.85 74.55 0.00 39.81 74.55 0.00 857.70 100.00 0.00 2.15 74.55 0.00 46.42 74.55 0.00 1000.00 100.00 0.00

[0387] In this experiment, the atomization time was 5 seconds per puff and the interval between puffs was 5 seconds. The transmittance, Dv(10), Dv(50) and Dv(90) were in an ups and downs cycle, which kept fluctuating with the atomization puffs.

[0388] 2. Mucosal irritation test of aerosol liquid composition in rats

[0389] 1) General observation of rats: During the administration period, rats in each group moved autonomously, drank water, and ate food. Their bowel movements and bowel movements were normal and their mental state was good. Their fur was shiny, and there was no abnormal secretion from the mouth, nose, or eyes. There was no abnormal breathing. No animals in each group died during the experiment. No obvious abnormalities were observed during the recovery period after drug withdrawal.

[0390] During the experiment, some animals in each group had congestion in their eyes. The abnormal animals basically recovered on the second day. This situation also occurred in the blank group, and there was no regularity. Analysis showed that it might be caused by compression of the animal cavity during the drug administration process and had nothing to do with the drug.

[0391] 2) White blood cell count and classification of bronchoalveolar lavage fluid: Administration period: Compared with the control group, there was no significant difference in the total white blood cell count of rats in the nebulized liquid composition nebulization administration group (P>0.05), and there was no significant difference in the percentage of neutrophils (NEUT%), lymphocytes (LYMPH%), eosinophils (EOS%), basophils (BASO%) and monocytes (MONO%) of rats in the nebulized liquid composition nebulization administration group (P>0.05). (The results are shown in Table 19). Recovery period: Compared with the control group, there was no significant difference in the total white blood cell count, neutrophil percentage (NEUT%), lymphocyte percentage (LYMPH%), eosinophils (EOS%), basophils (BASO%) and monocytes (MONO%) of rats in the nebulized liquid composition nebulization administration group (P>0.05). (The results are shown in Table 20).

[0392] Table 19 Effect of nebulized liquid composition on leukocytes in rat lung lavage fluid (drug administration period) n=8)

[0393]

[0394] Table 20 Effect of nebulized liquid composition on leukocytes in rat lung lavage fluid (recovery period) ( n=6)

[0395]

[0396] 3) Pathological observation: The results of gross observation showed that the local mucosal tissue of the respiratory tract of the animals in the nebulized drug administration group was similar to that in the control group, without congestion, edema, ulcers, etc. The results of histopathology showed that the effects of the nebulized liquid composition on the tracheal / bronchial epithelium, alveolar epithelium, nasal respiratory epithelium and olfactory epithelium were similar to those in the blank control group, with no obvious abnormalities. Typical pictures of male and female animals in the two groups are shown in Figure 16-19 . Fig.16 The blank control group was male. Fig.17 The blank control group was female. Fig.18 For the male group of the atomized liquid composition, Fig.19 The aerosol liquid composition group was female.

[0397] 3. Active systemic allergy test on guinea pigs by atomization inhalation of atomized liquid composition

[0398] 1) General condition: During the sensitization period, the guinea pigs in each group moved autonomously, drank water, and ate food. Their bowel movements and bowel movements were normal and their mental state was good. Their fur was shiny, and there was no abnormal secretion from their mouths, noses, and eyes, and no abnormal breathing was observed. No animal in each group died during the sensitization period.

[0399] 2) Body weight: Compared with the negative control group, there was no significant difference in the body weight of guinea pigs in the aerosol inhalation group during the experiment (P>0.05); (weight changes are shown in Table 21).

[0400] Table 21 Changes in body weight of guinea pigs after aerosol inhalation of aerosol liquid composition ( Sensitization n=8, stimulation n=4)

[0401]

[0402] 3) Allergic reaction: During the stimulation stage, the negative control group showed no allergic reaction symptoms, and the incidence of allergic reaction was 0. Guinea pigs in the positive control group showed allergic reactions such as urination, cyanosis, dyspnea and convulsion after stimulation, and eventually died, with an allergic reaction incidence of 100%. Animals in the drug administration group did not produce systemic allergic reactions such as piloerection, coughing and dyspnea after stimulation, and the incidence of allergic reaction was 0. The experimental results are shown in Table 22.

[0403] Table 22 Record of allergic reaction symptoms in guinea pigs after aerosol inhalation of aerosol liquid composition

[0404]

[0405] 4. Metabolic kinetics and tissue distribution of aerosol liquid composition

[0406] 4.1 Preliminary determination of chemical component concentrations in the atomized liquid composition

[0407] The results of the pharmacokinetic experiments after single and multiple administrations: Among the 10 components tested, three components, including quercetin-1, kaempferol, and diosmetin, could not be detected in plasma, and trace components were detected in the remaining 7 components: luteolin and its trace amount less than 0.1 ng / ml, acacetin and its trace components less than 0.1 ng / ml, luteolin-7-O-α-D-glucose anhydride, and diosmetin-7-O-α-D-glucose anhydride. tin-7-0-BD-glucopyranoside) and its trace components <0.1ng / ml, chlorogenic acid (Chlorogenicacid) can be detected and its trace components 0.1-3ng / ml, Buddleoside, and its trace components and trace components <0.1ng / ml, Eriocitrin (Eriocitrin) and its trace components <0.1ng / ml, Ferulic acid (Ferulicacid) can be detected as a trace component 0.1-6ng / ml.

[0408] Results of lung content: Quercetin-1, Kaempferol, Acacetin, Buddleoside, Eriocitrin, Diosmetin-7-0-BD-glucopyranoside were not detected, Luteolin and its trace amount were less than <0.5ng / ml, Diosmetin and its trace amount were <1ng / ml, Chlorogenicacid and its trace amount were <2ng / ml, and Ferulic acid and its trace amount were <3ng / ml.

[0409] 4.2 Results of tissue distribution study after nebulized inhalation of the nebulized liquid composition in rats

[0410] The concentrations of geraniol in the throat, trachea, lung, and plasma of rats at various time points after a single nebulization of the nebulizer liquid composition for 60 minutes are shown in Table 23; the concentrations of chlorogenic acid in the throat, trachea, lung, and plasma at various time points are shown in Table 24. The concentrations of luteolin-7-O-α-D-glucose anhydride in the throat, trachea, lung, and plasma at various time points are shown in Table 25, and the concentrations of luteolin in the throat, trachea, lung, and plasma at various time points are shown in Table 26; the concentrations of eriocitrin in the throat, trachea, lung, and plasma at various time points are shown in Table 27;

[0411] A small amount of luteolin, chlorogenic acid and trace amounts of luteolin-7-O-α-D-glucose anhydride, luteolin and erythritol were detected in the throat, trachea and lungs of rats, while the above five components were not detected in plasma.

[0412] Table 23 Concentration of dianthrin in throat, trachea, lung and plasma at different times (ng / mL)

[0413]

[0414] Table 24 Concentration of chlorogenic acid in plasma, lung, trachea and brain at different time points (ng / mL)

[0415]

[0416]

[0417] Table 25 Concentration of luteolin-7-O-α-D-glucose anhydride in throat, trachea, lung and plasma at each time point (ng / mL)

[0418]

[0419] Table 26 Concentration of luteolin in throat, trachea, lung and plasma at each time point (ng / mL)

[0420]

[0421]

[0422] Table 27 Concentration of eriocitrin in throat, trachea, lung and plasma at each time point (ng / mL)

[0423]

[0424] IV. Conclusion

[0425] 1. Study on atomized particle size of atomized liquid composition

[0426] The atomization particle size distribution results of the atomized liquid composition in this experiment are as follows: Dv(10)=0.4μm, Dv(50)=0.5337μm, Dv(90)=345μm, and transmittance=14.6%. Among them, Dv(10), Dv(50), and Dv(90) are the droplet particle sizes corresponding to the cumulative volume fractions of the aerosol of 10%, 50%, and 90%, respectively. The median particle size Dv(50) is the most commonly used parameter for evaluating the aerosol particle size distribution. In terms of its particle size distribution, the atomization median particle size of the atomized liquid composition is 0.5337μm. Generally speaking, the larger the aerosol particle size, the easier it is to deposit in the upper respiratory tract or even the extrathoracic area. When exhaling, aerosols with smaller diameters can be quickly exhaled, with particle sizes as small as 0.5μm. At present, many studies have shown that during normal tidal breathing, the atomized aerosol particle size of 1 to 5μm is the optimal range for pulmonary administration. The atomized particle size of the atomized liquid composition of the present invention is conducive to entering the blood through the lungs to exert pharmacological effects.

[0427] 2. Mucosal irritation test of aerosol liquid composition in rats by aerosol inhalation

[0428] Continuous nebulization of the nebulized liquid composition for 60 minutes and continuous nebulization inhalation for 7 days had no significant effect on the general condition of the rats, no significant effect on the leukocyte count and classification of the lavage fluid, and no abnormal changes were observed during the recovery period. Histopathology showed that the histopathological changes in the oral cavity, nose, pharynx, trachea, lungs, etc. of the administration group were similar to those of the control group, with no significant changes. The analysis showed that the nebulized liquid composition was administered by nebulization inhalation using a matching nebulizer device without irritation to the rats.

[0429] 3. Active systemic allergy test on guinea pigs by atomization inhalation of atomized liquid composition

[0430] The test used an active systemic allergy test for guinea pigs, and oral and nasal aerosol inhalation was used for both the sensitization and the stimulation stage. The guinea pigs were observed to have allergic reactions by sensitization three times a day and stimulation with a double dose. The guinea pigs were generally in good condition and had normal weight gain during the sensitization and stimulation of the aerosol liquid composition. The stimulation did not cause allergic reactions in the guinea pigs. The analysis showed that the aerosol liquid composition did not cause allergic reactions in guinea pigs when it was aerosolized using a matching aerosol device.

[0431] 4. Metabolic kinetics and tissue distribution of aerosol liquid composition

[0432] 4.1 Preliminary determination of chemical component concentrations in the atomized liquid composition

[0433] In this experiment, the ingredients in the nebulized liquid composition were first analyzed and tested, and quercetin, luteolin, kaempferol, acacetin, dioscorea lignin, chlorogenic acid, ferulic acid, luteolin-7-O-α-D-glucose anhydride, and montmorillonine were determined as content determination components based on the prescription composition. The results of single and multiple plasma tests at different time points showed that very few components entered the blood after nebulized inhalation, and the concentrations of the eight components that could be detected were mostly below 0.1ng / ml. The quantitative detection line of this determination was 0.1ng / ml, which was lower than the minimum detection line of this test. Therefore, there was a certain error in the plasma content after single and multiple administrations, and no regular changes were observed at different time points.

[0434] In this experiment, the lung tissue was tested 24 hours after administration by nebulization inhalation. Among the 10 components tested, 4 components could be detected within 24 hours: luteolin concentration was less than <0.5ng / ml, geraniol concentration was <1ng, chlorogenic acid concentration was <2ng / ml, and ferulic acid concentration was <3ng / ml. The lung tissue was tested 24 hours after administration, but its content was still greater than that in the blood, indicating that after administration by nebulization inhalation, the drug was concentrated in the local lungs and its drug concentration was higher than the systemic blood concentration.

[0435] 4.2 Pharmacokinetics and tissue distribution studies in rats after administration of the nebulized liquid composition

[0436] The results of the pharmacokinetic experiment showed that among the five components tested, only erythrocitroside could be detected in trace amounts at certain time points, and the other four components were not detected at any time point, and the pharmacokinetic parameters could not be calculated. The tissue distribution results showed that a small amount of diosmetin, chlorogenic acid and trace amounts of luteolin-7-O-α-D-glucose, luteolin, and erythrocitroside could be detected in the throat, trachea, and lungs of rats, and the five components were not detected in plasma. Among the five components tested, diosmetin and chlorogenic acid were not detected in the throat, trachea, lungs, and plasma 7 days after administration, and luteolin-7-O-α-D-glucose, luteolin, and erythrocitroside were not detected in the throat, trachea, lungs, and plasma 24 hours after administration. This suggests that its components do not remain in the respiratory tract. The concentrations of the five substances in the throat, trachea, lungs, and blood are: throat, trachea>lung>plasma. After atomization, the concentration of the atomized liquid composition in the throat, trachea, lungs and blood decreases in sequence, and enters the blood from the lungs to exert its effect. 24 hours after atomization administration, no active ingredient was detected in the throat, trachea, lungs and plasma, indicating that the atomized liquid composition did not remain in various tissues 24 hours after atomization inhalation.

Claims

1. An atomized liquid composition, characterized in that: The atomized liquid composition is prepared from the following components in parts by mass: 。 2. The atomized liquid composition according to claim 1, characterized in that: The atomized liquid composition is prepared from the following components in parts by mass: 。 3. The atomized liquid composition according to claim 1, characterized in that: The mass fraction of the chrysanthemum concentrate and the lemon concentrate is 5-25%; preferably, the mass fraction of the chrysanthemum concentrate and the lemon concentrate is 10%.

4. The atomized liquid composition according to claim 3, characterized in that: The mass ratio of the chrysanthemum concentrate to the lemon concentrate is 3-5:

1.

5. The atomized liquid composition according to claim 1-2, characterized in that: The sweetener is selected from one or more of neotame, mogroside and steviol glycoside.

6. The atomized liquid composition according to claim 1-2, characterized in that: The cooling agent is selected from one or more of menthol, menthone, mint extract, menthyl acetate, N,2,3-trimethyl-2-isopropylbutyramide, and natural menthol.

7. The atomized liquid composition according to claims 1-3, characterized in that: The chlorogenic acid content in the atomized liquid composition is 0.1-1 mg / mL.

8. The atomized liquid composition according to claims 1-3, characterized in that: The chrysanthemum concentrate is prepared according to the following method: Weigh chrysanthemum, add 80% (V / V) ethanol aqueous solution, heat and reflux for extraction, filter and take filtrate; add 80% (V / V) ethanol aqueous solution to the residue, heat and reflux for extraction, filter, combine the two filtrates, concentrate the filtrate under reduced pressure at 42° C. to 45° C. to contain 0.95 to 1.05 g of crude drug per milliliter, let the concentrated solution cool to room temperature, filter or centrifuge to remove solids, take the clear solution, add propylene glycol for dilution, and obtain a concentrated solution with a crude drug concentration of 0.3 g / mL.

9. The method for preparing the atomized liquid composition according to claims 1-4, characterized in that: The following steps are involved: A. Weigh the formulated amount of propylene glycol into a container, heat it and add the sweetener to mix well; add glycerin and mix well; B. Accurately weigh the amount of purified water, add it into the container, and mix well; C. Accurately weigh the cooling agent in the formula and add it into the container and mix well; D. Add chrysanthemum concentrate and lemon concentrate into the above container and mix well; after the mixed solution cools to room temperature, store it for filling the atomizer.

10. An atomizing device, characterized in that: The atomization device comprises an atomization rod and an atomizer, wherein a control panel, a battery, an indicator light, an airflow sensor and a charging interface are arranged inside the atomization rod; the atomizer comprises a heating core, a liquid storage chamber, an airflow channel and a suction nozzle; the suction nozzle is connected to the airflow channel, the air pressure sensing channel and the air inlet in sequence; the airflow sensor is used to detect the suction negative pressure of the air pressure sensing channel and transmit the signal to the control panel, the control panel receives the signal and drives the battery to supply power to the heating core, and the heating core heats and atomizes the atomized liquid composition in the liquid storage chamber into an aerosol.

11. The atomizing device according to claim 10, characterized in that: The heating core is a ceramic core with a heating power of 3-4.5 watts, an open porosity of 54±4%, and a water absorption rate of 50±5%.

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