Preparation method of anti-chronic pharyngitis microcapsule and application of anti-chronic pharyngitis microcapsule in tobacco products

Abstract

The invention discloses a preparation method of an anti-chronic pharyngitis microcapsule and application of the anti-chronic pharyngitis microcapsule in tobacco products, the anti-chronic pharyngitis microcapsule is composed of a core material and a wall material, the wall material comprises gelatin and Arabic gum, the core material comprises tea tree essential oil, (+)-borneol and ionic liquid, and the ionic liquid is prepared by reaction of vanillin and choline bicarbonate. The natural anti-inflammatory component tea tree essential oil and (+)-borneol serving as an absorption enhancer and having an anti-inflammatory effect are encapsulated in an ionic liquid microemulsion system through a complex coacervation method, the water solubility and bioavailability of the tea tree essential oil and the (+)-borneol are remarkably improved, and the solubility and the transdermal absorption effect of the medicine are effectively improved. The key medicinal components in the microcapsules can be effectively transferred into mainstream smoke aerosol, and compared with a cut tobacco adding mode, the heating non-combustion cigarette filter stick hollow cooling section adding mode is more beneficial to transfer of the key medicinal components to mainstream smoke.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco flavors, and in particular to a preparation method of anti-chronic pharyngitis microcapsules and application thereof in tobacco products. Background Art

[0002] Tobacco and health issues currently occupy a pivotal position in the tobacco industry. Cigarettes, while inhaled, can provide consumers with a sense of satisfaction and pleasure, the long-term effects of smoke on receptors in the throat and other areas can lead to conditions such as chronic pharyngitis. Currently, the incidence of chronic pharyngitis is relatively high among smokers. Given the high prevalence of chronic pharyngitis among smokers and the growing public health awareness, the tobacco industry and companies are actively seeking innovative approaches, focusing on developing new cigarettes with ingredients that are beneficial for throat protection, in order to effectively reduce the risk of throat diseases.

[0003] Currently, antibiotics are the first choice for treating pharyngitis, but long-term use of antibiotics can cause side effects such as diarrhea, nausea, vomiting, rash and drug resistance. Abuse can lead to dysbiosis of the throat flora, causing dual infection, and the patient's condition may even worsen. Traditional Chinese medicine has attracted much attention due to its precise efficacy, small side effects and therapeutic potential, but it has poor water solubility and low bioavailability, making it difficult to directly apply for inhalation. Existing microencapsulation technology has low encapsulation efficiency for volatile components and insufficient stability. Traditional microemulsion preparation methods are difficult to encapsulate multiple fat-soluble components at the same time, and the process is complicated (such as high temperature and high pressure). Free drugs are easily lost due to volatility when directly administered, making it difficult to release them continuously at the lesion site.

[0004] Therefore, it is of great practical significance to combine the theory of traditional Chinese medicine with the development experience of tobacco flavors to develop tobacco flavors that have both sensory enjoyment and medicinal value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of anti-chronic pharyngitis microcapsules and their application in tobacco products, which solves the technical difficulties of poor water solubility, low bioavailability and low transfer rate to mainstream smoke of tea tree essential oil and (+)-borneol in traditional tobacco addition methods, and realizes the effective application of high-efficiency anti-inflammatory ingredients in tobacco products.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] The invention discloses an anti-chronic pharyngitis microcapsule, which is composed of a core material and a wall material. The wall material comprises gelatin and gum arabic, and the core material comprises tea tree essential oil, (+)-borneol and an ionic liquid. The ionic liquid is prepared by reacting geranic acid with choline bicarbonate.

[0008] Preferably, in the above technical solution, the molar ratio of geranic acid to choline bicarbonate is 2:1, the mass ratio of tea tree essential oil, (+)-borneol and ionic liquid is 1:1:2, the mass ratio of core material to wall material is 1:2, and the mass ratio of gelatin to gum arabic is 1:1.

[0009] A method for preparing microcapsules for treating chronic pharyngitis comprises the following steps:

[0010] (1) Preparation of ionic liquid: Geranic acid and choline bicarbonate were dissolved in methanol respectively, the two solutions were mixed and stirred, methanol and water generated by the reaction were removed by rotary evaporation, and the ionic liquid was obtained by vacuum drying;

[0011] (2) Preparation of microemulsion: Tween-80 was dissolved in anhydrous ethanol to prepare an emulsifier solution, and the solution was ultrasonicated at room temperature for 5 minutes and then vortexed for 2 minutes until the solution was clear and transparent, indicating complete dissolution; tea tree essential oil and (+)-borneol were dissolved in ionic liquid and ultrasonicated until there were no solid particles and the solution was clear and transparent, and then the emulsifier solution was added to form a mixed solution, which was fully mixed as the oil phase. The oil phase was added dropwise to the aqueous phase until the solution changed from clear to turbid, and the addition was stopped. The solution was stirred and ultrasonicated to obtain a microemulsion;

[0012] (3) Preparation of microcapsules: Gum arabic and gelatin were dissolved in water and mixed to obtain a composite wall material solution, which was added to a microemulsion. The pH was adjusted to 4.0 with 1% glacial acetic acid and stirred to form an emulsion. The pH was then adjusted to 6.0 with NaOH. TG enzyme was added to fix the microcapsule structure. The microcapsule solution was obtained after stirring for 6 hours. The ethanol and unencapsulated tea tree essential oil and (+)-borneol were removed by rotary evaporation. The remaining solution was collected as a microcapsule solution and vacuum freeze-dried to obtain microcapsule powder.

[0013] Preferably, in the above technical solution, in step (1), the molar ratio of geranic acid to choline bicarbonate is 2:1, the stirring time is 1 day, the rotary evaporation temperature is 40°C to remove methanol, 60°C to remove water, and the drying time is 48 hours.

[0014] Preferably, in the above technical solution, in step (2), the mass ratio of tea tree essential oil: ionic liquid is 1:2, the mass ratio of emulsifier Tween-80 and co-emulsifier anhydrous ethanol is 3:1, and the mass ratio of tea tree essential oil and emulsifier is 3:1.

[0015] Preferably, in the above technical solution, in step (3), the mass ratio of the core material to the wall material is 1:2, the mass ratio of gelatin to gum arabic is 1:1, and the complex coagulation pH is 4.0.

[0016] The invention discloses an application of an anti-chronic pharyngitis microcapsule in the preparation of a heat-not-burn tobacco product, wherein the microcapsule is added to the heat-not-burn tobacco product in an amount of 9.3-18.6 mg / piece.

[0017] Preferably, in the above technical solution, in the heat-not-burn tobacco product, the microcapsules are added to the hollow cooling section of the filter rod, and the material of the hollow cooling section is polypropylene fiber.

[0018] Application of an anti-chronic pharyngitis microcapsule in the preparation of an anti-chronic pharyngitis drug.

[0019] Preferably, in the above technical solution, the dosage form of the drug is a spray, lozenge or pill.

[0020] The above technical solution of the present invention has the following beneficial effects:

[0021] The present invention discloses a preparation method and application of tea tree essential oil and (+)-borneol ionic liquid microcapsules. The natural anti-inflammatory ingredient tea tree essential oil and (+)-borneol as an absorption enhancer with anti-inflammatory effect are encapsulated in an ionic liquid microemulsion system by a complex coacervation method. This not only significantly improves the water solubility and bioavailability of tea tree essential oil and (+)-borneol, but also effectively improves the solubility and transdermal absorption effect of the drug. The key active ingredients in the microcapsules can be effectively transferred to the mainstream smoke aerosol. Compared with the tobacco shred addition method, the heat-not-burn cigarette filter rod hollow cooling section addition method is more conducive to the transfer of key active ingredients to the mainstream smoke. In addition, animal experiments have shown that the microcapsules can effectively inhibit inflammatory factors such as IL-6, IL-1β, TNF-α, repair throat mucosal damage, and are highly safe and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0023] Figure 1 This is the GC-MS chart of tea tree essential oil.

[0024] Figure 2 This is the hydrogen spectrum of ionic liquid.

[0025] Figure 3 This is the infrared spectrum of ionic liquid.

[0026] Figure 4 These are the results of characterization of the particle size and potential of microemulsions, including: A. Particle size variation of microemulsions of different groups over time; B. Particle size distribution of microemulsions with the most suitable prescription; C. Particle potential variation of microemulsions with the most suitable prescription over time.

[0027] Figure 5 Characterization diagram of the particle size and potential stability of the microcapsules, among which, D. microcapsule particle size change with time; E. microcapsule potential change with time.

[0028] Figure 6This is the standard curve of 4-terpineol and terpinene in tea tree essential oil.

[0029] Figure 7 This is the standard curve of (+)-borneol.

[0030] Figure 8 For pre-experimental laryngoscope.

[0031] Figure 9 H&E staining for pre-experimental purposes.

[0032] Figure 10 Figure 3 is a graph showing changes in mouse body weight.

[0033] Figure 11 The blood routine test results of rats in each group are shown in Figure 2 (**, *** indicate p<0.01, p<0.001, respectively, compared with the blank group. #, ##, ### indicate p<0.05, p<0.01, p<0.001, respectively, compared with the model group. The rest showed no significant differences).

[0034] Figure 12 Blood biochemical graphs of rats in each group (*, *, respectively indicate p<0.05 and p<0.001 compared with the blank group).

[0035] Figure 13 H&E staining of the liver (magnification 20 times).

[0036] Figure 14 H&E staining of the lungs (magnification 20 times).

[0037] Figure 15 H&E staining of kidney (magnification 20 times).

[0038] Figure 16 These are the laryngoscope observation pictures of each group.

[0039] Figure 17 This is an H&E staining observation picture of the throat.

[0040] Figure 18 Graph showing serum inflammatory factor levels (*, ** indicate p<0.05, p<0.01 compared with the blank group, respectively. ##, ### indicate p<0.05, p<0.01, p<0.001 compared with the model group, respectively. The rest showed no significant differences).

[0041] Figure 19 is the transfer rate of key active ingredients of microcapsules in mainstream smoke. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention.

[0043] Example

[0044] (1) Experimental materials:

[0045] (1) Animals: SPF-grade SD rats (half male and half female, weighing 180-220 g, 10 weeks old) were purchased from Shandong Jinan Pengyue Experimental Animal Breeding Co., Ltd. They were acclimated to the rats without food or water restriction for 2 weeks. Afterwards, the rats were modeled and administered with drugs.

[0046] (2) Drugs and reagents: Tea tree essential oil, Elaine Biotechnology Co., Ltd.; (+)-Borneol (Cat. No. B119291), Shanghai Aladdin Biochemical Technology Co., Ltd.; Geranic acid (Cat. No. A18750), China Alfa Aesar Chemical Co., Ltd.; Choline bicarbonate (Cat. No. C7519), Shanghai Sigma-Aldrich Trading Co., Ltd.; Anhydrous methanol (Tianjin Fuyu Fine Chemical Co., Ltd.), Anhydrous ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.), Acetonitrile (HPLC grade), Shanghai Honeywell Trading Co., Ltd., Shuyanqing spray (Guilin Sanjin Pharmaceutical Co., Ltd.),

[0047] (3) Instruments: 1 / 10,000 electronic analytical balance (METTLER TOLEDO), rotary evaporator (Zhengzhou Great Wall Science & Technology Co., Ltd.), low-temperature coolant circulation pump (Zhengzhou Great Wall Science & Technology Co., Ltd.), ultrapure water machine (Shanghai Sigma-Aldrich Trading Co., Ltd.), vacuum drying oven (Henan Jielong Technology Co., Ltd.), integrated ultrasonic processor (Shanghai Chongfeng Scientific Instrument Co., Ltd.), particle size-potential analyzer (Brookhaven Instrument Company, USA), high performance liquid chromatograph (Agilent Technologies, USA), constant temperature water bath (Shanghai Yiheng Scientific Instrument Co., Ltd.), pH meter (Shanghai Yidian Scientific Instrument Co., Ltd., China).

[0048] (2) Experimental methods:

[0049] (1) Qualitative and quantitative analysis of tea tree essential oil

[0050] Tea tree essential oil samples were mixed with dichloromethane and filtered through a 0.22 μm microporous membrane. Gas chromatography-mass spectrometry (GC-MS) was used for qualitative and quantitative analysis of tea tree essential oil.

[0051] GC-MS instrument conditions were: carrier gas flow rate 1.0 mL / min; injection volume 1 μL; solvent delay 2.1 min. Inlet temperature 280°C; column oven initial temperature 60°C, ramped to 280°C at 10°C / min; retention time 5 min. Split ratio 100:1, flow rate 100 mL / min. Ionization mode: EI, ionization energy: 70 eV. Ion source temperature 230°C, quadrupole temperature 150°C, scan range (m / z): 10–550 amu.

[0052] (2) Preparation and characterization of ionic liquids

[0053] Geranic acid and choline bicarbonate were measured and dissolved in methanol for reaction. 3596 μL (0.02 moL) of geranic acid was dissolved in 2 mL of methanol. 1412 μL (0.01 moL) of choline bicarbonate was also dissolved in 1 mL of methanol. The mixture was then placed in a 25 mL round-bottom flask and stirred magnetically. The geranic acid methanol solution was then added dropwise to the 25 mL round-bottom flask while stirring. The molar ratio was 2:1.

[0054] The mixture solution was stirred at room temperature for 1 day, and then the methanol was removed by rotary evaporation at 40°C, and then the water generated by the reaction was removed by rotary evaporation at 60°C, and then vacuum dried for 48 hours to obtain ionic liquids (CAGE ILs).

[0055] By observing the ionic liquid morphology, hydrogen spectrum and infrared, it can be judged whether the ionic liquid is successfully prepared.

[0056] (3) Preparation of Tea Tree Essential Oil-(+)-Borneol Ionic Liquid Microemulsion

[0057] First, prepare an emulsifier solution, i.e., an emulsifier + a co-emulsifier (the emulsifier is Tween-80, and the co-emulsifier is ethanol). Weigh an appropriate amount of Tween-80 and dissolve it in anhydrous ethanol to prepare the solution. Ultrasound for 5 minutes to promote dissolution, then vortex for 2 minutes until the solution is clear and transparent, indicating complete dissolution. Weigh an appropriate amount of tea tree essential oil and (+)-borneol and dissolve them in the ionic liquid. Ultrasound dissolution is performed until there are no solid particles and the solution is clear and transparent. Then, the emulsifier solution is added to form a mixed solution, and thoroughly mixed to form the oil phase.

[0058] 10 mL of water was placed in a round-bottom flask on a magnetic stirrer to form the aqueous phase. The oil phase was added dropwise to the aqueous phase while stirring (addition continued after the solution stabilized). Addition was stopped when the solution turned from clear to turbid, and stirring was continued for 6 hours. Ultrasonic probe-assisted dispersion was used to terminate the reaction to obtain an emulsion. A blank microemulsion was also prepared, containing only the ionic liquid microemulsion but without tea tree essential oil or (+)-borneol, and was prepared using the same method.

[0059] (4) Preparation of Tea Tree Essential Oil-(+)-Borneol Ionic Liquid Microcapsules

[0060] Appropriate amounts of gum arabic and gelatin as wall materials are dissolved in water and mixed to form a composite wall material solution. This is then added to a microemulsion, the pH adjusted with 1% glacial acetic acid, and stirred at room temperature for 6 hours to form an emulsion. The pH is then adjusted with sodium hydroxide, and TG enzyme is added to fix the microcapsule structure. Stirring is continued for another 6 hours to obtain a microcapsule solution. Ethanol, unencapsulated tea tree essential oil, and borneol are removed by rotary evaporation at 40°C. The remaining solution is then collected to form the microcapsule solution. Vacuum freeze-drying is then performed to obtain a microcapsule powder.

[0061] (5) Characterization of microcapsules

[0062] 1) Particle size, zeta potential and morphology analysis: An appropriate amount of the prepared microcapsule solution was taken and passed through a 0.22 μm filter membrane. The filtrate was then used to determine the particle size distribution and zeta potential of the nanoparticles using a particle size potential analyzer.

[0063] 2) Determination of encapsulation efficiency and drug loading

[0064] First, a standard curve for tea tree essential oil and (+)-borneol was prepared. A gradient concentration series of dilute ethanol solutions of tea tree essential oil was prepared. These solutions were squeezed through a 0.22 μm organic filter membrane and placed in an HPLC injection vial. The peak area at a wavelength of 190 nm was measured by high-performance liquid chromatography, and a linear regression curve was plotted. A series of (+)-borneol dilute ethanol solutions with mass concentrations of 10, 20, 40, 80, 100, 200, and 400 μg / mL were prepared. These solutions were squeezed through a 0.22 μm organic filter membrane and placed in an HPLC injection vial. The peak area at a wavelength of 190 nm was measured by high-performance liquid chromatography, and a linear regression curve was plotted.

[0065] To determine the drug encapsulated in microcapsules, first destroy the microcapsules, take 2 mL of microcapsules in parallel and add 4 mL of ethanol, sonicate for 20 minutes (add ice cubes to prevent the loss of volatile substances due to excessive temperature), centrifuge at 1000 rpm for 3 minutes, take the supernatant, squeeze it through a 0.22 μm organic filter membrane, and place it in an HPLC injection bottle.

[0066] Tea tree essential oil was determined using a Shim-pack GIST C18 column (25 mm × 4.6 mm, 5 μm), a mobile phase of acetonitrile:water (70:30), a column thermostat at 25°C, an injection volume of 1 μL, and a flow rate of 1.0 mL / min. The peaks at retention times of 6.670 and 13.070 min were detected. (+)-Borneol was determined using a C18 column, a mobile phase of acetonitrile:water (60:40), a column thermostat at 25°C, an injection volume of 1 μL, and a flow rate of 1.0 mL / min. The peak at retention time was 8.190 min.

[0067] Take 2.0 mL of the prepared microcapsules, add 4 mL of ethanol, and ultrasonicate for 20 min (control the temperature to prevent the loss of some volatile substances due to excessive temperature), destroy the microcapsules, and take the supernatant. The amount of drug encapsulated in the nanoparticles (M 包 ), the encapsulation efficiency can be calculated. At the same time, the upper nanoparticles were freeze-dried and weighed (M 总 ), calculate the drug loading amount.

[0068] Encapsulation efficiency = M 包 / M 投药量

[0069] Drug loading = M 包 / M 总

[0070] 3) Optimization of microcapsule preparation process: Conduct single factor or orthogonal experimental investigations on factors that may affect microcapsule quality during the preparation process, such as the ratio of choline geranyl, emulsifier dosage, core-to-wall ratio, wall material ratio, and pH. Determine the optimal process based on particle size and zeta potential.

[0071] 4) Stability study: The microcapsule solution was placed at room temperature for 14 days, and the changes in the nanoparticle size, PDI and Zeta potential were detected.

[0072] (6) Determination of the transfer rate of key components of tea essential oil in mainstream smoke

[0073] To reduce the impact of the drug components in the microcapsules on the sensory quality of cigarettes, preliminary experiments were conducted at a minimum effective dose of 5 mg / mL. Based on the interspecies dose conversion coefficient of rat:human = 6.3:1, the human equivalent dose was calculated to be 2.65 mg / kg. Based on a standard adult weight of 70 kg, the theoretical daily dose should be 185.5 mg. Based on the daily consumption of 20 cigarettes, the injection dose per cigarette is estimated to be 9.3 mg / cigarette.

[0074] The prepared microcapsules were dispersed in an ethanol solution and then injected into a certain brand of cut tobacco at a concentration of 9.3 mg / cigarette. The cigarette samples were equilibrated under the conditions specified in GB / T 16447-2004 and then smoked according to the method specified in GB / T 19609-2004. Ten cigarettes of each type were divided into two groups. TPM from the mainstream smoke of five cigarettes was collected using each Cambridge filter disc. After smoking, five empty puffs were taken to allow the mainstream smoke to settle freely for 30 seconds. The Cambridge filter disc was then removed and placed in a stoppered Erlenmeyer flask. 18 mL of extraction solution was added, and ultrasonic extraction was performed at room temperature for 30 minutes. The volume was then adjusted to 25 mL using the extraction solution and filtered through a 0.45 μm organic phase microporous membrane for the sample to be tested.

[0075] (7) Animal experiments on microcapsules against chronic pharyngitis

[0076] 1) Pharyngitis Model Establishment: Model-inducing drug: 2.5% ammonia solution. The model group was administered 2.5% ammonia solution, with the drug sprayed into the rat pharynx using a laryngeal applicator, twice daily (10:00 AM and 4:00 PM, 6 hours apart), three times per session, for 3 weeks. A blank group was administered an equal amount of pure water as a normal control. Behavioral observations were conducted during this period. 28 days after modeling, the rats were fasted for 12 hours and then intraperitoneally injected with an anesthetic. Once the rats were anesthetized, the larynx was examined using a visual laryngoscope and photographed for analysis. Blood was then drawn from the abdominal aorta, and a small amount was used for routine blood tests. Pharyngeal tissue was removed and fixed with 4% formaldehyde for 3 days, followed by decalcification in EDTA solution for 30 days (the solution was changed every 2 days). Pathological sections were then stained with hematoxylin and eosin to observe morphological changes in the pharyngeal tissue. Successful modeling was evaluated.

[0077] 2) Animal Experiment Grouping: 72 Sprague-Dawley rats, half male and half female, weighing 250-300g were selected for the study. They were housed in a clean animal laboratory at a temperature of 18-22°C with adequate food and water. After one week of adaptive feeding, all rats, except the blank group, were given ammonia. After model establishment, the remaining rats, excluding the blank group, were randomly divided into a model group, a positive drug group (Sanjin Shuyanqing spray, diluted 1 / 2), a tea tree essential oil group (1 mg / mL), a (+)-borneol group (1 mg / mL), a tea tree essential oil + (+)-borneol group (500 + 500 μg / mL), and a tea tree essential oil + (+)-borneol ionic liquid microcapsule group (low, medium, and high concentrations, 500, 750, and 1000 μg / mL, equivalent to the drug concentrations of the essential oil and borneol), with 8 rats per group, for a total of 9 groups, totaling 72 rats. The model and blank groups were given equal amounts of purified water.

[0078] 3) Animal Dosing: Throat spray administration, twice daily (9:00 AM and 4:00 PM, 8 hours apart), 4 doses / time, for 21 days. Body weights were measured every other day during the dosing period.

[0079] 4) In vivo toxicity studies: During the dosing period, changes in body weight, diet, and behavior of the rats in each group were recorded. Heart, liver, spleen, lung, kidney, and testicles were isolated and stained with H&E. The results were compared with those of the normal group and the positive drug group to observe any significant pathological changes.

[0080] 5) Throat Characteristics: On the last night of dosing, rats were kept refraining from eating or drinking. Anesthesia was administered intraperitoneally. Immediately after anesthesia, the rats' larynxes were examined using a visual laryngoscope and photographed for analysis.

[0081] 6) Blood biochemistry, routine blood test, and inflammatory factor testing: Blood was collected from the abdominal aorta. A small amount of whole blood was collected for routine blood test. The remaining blood was centrifuged at 3500 rpm for 10 minutes to obtain rat serum, which was aliquoted and stored at -80°C. Biochemical markers, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6), were assayed using ELISA kits.

[0082] 7) Pharmacodynamic Study: Rat pharyngeal tissue was removed and weighed. The tissue was first fixed with 4% formaldehyde for 3 days, with the solution changed daily. The tissue was then fixed with EDTA decalcification solution for 30 days (with the solution changed every 2 days). Pathological sections were then stained with H&E to observe pathological changes in the pharyngeal tissue.

[0083] (8) Statistical analysis

[0084] GraphPad Prism 8 statistical software was used to analyze and process the data. Data for each group are expressed as mean ± standard deviation (x ± s). Multiple groups were compared using one-way analysis of variance, and pairwise comparisons were performed using the LSD test. Differences were considered statistically significant when P < 0.05.

[0085] (3) Experimental results:

[0086] (1) Qualitative and quantitative analysis of tea tree essential oil

[0087] GC-MS results are as follows Figure 1 It can be seen that 4-terpineol (41.68%), γ-terpinene (11.88%), m-isopropyl toluene (6.85%), (+)-4-carene (5.92%), and α-terpineol (4.61%) are its main components, which is consistent with existing literature data.

[0088] Table 1 Tea tree essential oil components and content

[0089]

[0090]

[0091] (2) Appearance and characterization of ionic liquids

[0092] The ionic liquid is a yellow, clear, and transparent liquid with a certain degree of fluidity and viscosity. It also maintains good fluidity and does not freeze when placed in a -20°C refrigerator.

[0093] Figure 2 For the hydrogen spectrum ( 1H-NMR), the position of geranic acid at 11.90 ppm is -COOH peak, and geranic acid-related peaks appear at chemical shift values ​​of 5.0-5.60 ppm and 1.58-2.64 ppm in the hydrogen spectrum of the product ionic liquid. At the same time, the -COOH peak disappears, and a choline bicarbonate-related peak appears at 3.40-3.86 ppm. It is preliminarily judged that the preparation is successful.

[0094] Figure 3 Infrared spectrum (FT-IR), in the FT-IR spectrum of ionic liquid, 3222cm -1 The -OH stretching vibration peak of the carboxyl group of choline bicarbonate is significantly weakened because it participates in the reaction; -1 、2914cm -1 、2856cm -1 They are -CH3, -CH2, and -CH stretching vibration peaks of geranic acid; 1645 cm -1 The C=C stretching vibration peak of geranic acid; 1478 cm -1 It is the alcohol-OH stretching vibration peak of choline bicarbonate.

[0095] Based on the appearance, hydrogen spectrum and infrared spectrum of the ionic liquid, it was determined that the ionic liquid was successfully prepared.

[0096] (3) Preparation and characterization of microcapsules

[0097] 1) Preparation and Characterization of Microemulsions: The optimal ratio was determined based on particle size and room temperature stability. The three factors were: essential oil:ionic liquid, Km (emulsifier:co-emulsifier), and essential oil:emulsifier mass. A three-factor, three-level orthogonal experiment was conducted as follows.

[0098] Table 2 Three factors and three levels of microemulsion

[0099]

[0100]

[0101] Table 3 Microemulsion orthogonal experiment table

[0102]

[0103] Table 4 Particle size and PDI of each group in orthogonal experiment

[0104]

[0105] In addition, a blank microemulsion was prepared with a particle size of 52.22±1.20nm. The particle size of the drug-loaded group was larger than that of the blank group, indicating that the drug was loaded to a certain extent. Groups 1, 5, and 7 were selected for further screening to continue observing the changes in particle size, i.e., stability. Figure 4Figure A shows the particle size changes over a week in Groups 1, 5, and 7, with time as the horizontal axis and particle size as the vertical axis. All groups remained very stable on Day 7, with no stratification or precipitation. Particle size was 140.43 ± 2.09 nm, with values ​​of 1 < 7 < 5. Furthermore, the PDI of Group 1 was smaller than that of the other groups, indicating a more uniform particle size. The microemulsion potential of each group was positive and greater than 10, showing no significant change.

[0106] Based on the changes in particle size and potential, it can be judged that the microemulsion is very stable. Finally, a preparation method was selected as the optimal method, namely the optimal conditions: essential oil: ionic liquid = 1:2, Km = 3:1, essential oil: emulsifier = 3:1.

[0107] 2) Preparation and Characterization of Microcapsules: The most appropriate ratio was determined based on particle size and room temperature stability. The three factors were: core-to-wall ratio, wall material ratio, and complex coagulation pH. A three-factor, three-level orthogonal experiment was conducted, as shown in the following table:

[0108] Table 5 Microcapsules three factors three levels

[0109]

[0110] Table 6 Microcapsule orthogonal experiment table

[0111]

[0112] Potential measurements revealed that the microcapsule potential was negative, while the microemulsion potential was positive, indicating a potential reversal. This is because the gum arabic used in the complex coacervation method has a high negative charge, while gelatin has a positive charge at a certain pH. The gum arabic contains some unreacted anions, so the microcapsule potential should be negative. This indicates, to a certain extent, that the drug is encapsulated.

[0113] As storage time at room temperature progressed, some groups began to precipitate, indicating instability. Based on a comprehensive analysis of particle size and PDI, group 8 was selected as the optimal condition, with time in days as the horizontal axis and particle size as the vertical axis. In the figure, the particle size was 239.62 ± 8.31 nm and the PDI was 0.233 ± 0.012. After one week, the particle size was 201.23 ± 1.58 nm and the PDI was 0.224 ± 0.007, indicating that the microcapsules were relatively stable.

[0114] 3) Microcapsule Encapsulation Efficiency and Drug Loading: Based on the standard curves for tea tree oil and (+)-borneol, the encapsulation efficiencies of tea tree oil and (+)-borneol in the microcapsules were (82.26±3.01)% and (82.81±2.33)%, respectively. The encapsulation efficiency of (+)-borneol was (69.75±2.13)%. The final calculated drug loadings were 5.6% for the essential oil and 4.4% for the borneol.

[0115] (4) Animal experiment results

[0116] 1) Chronic pharyngitis model

[0117] During the modeling period, the rats in the model group experienced increased water intake and decreased food intake, mental fatigue, coarse and dark fur, increased urine output, oral ulcers in some rats, and even death. Laryngoscopic observation revealed that the throats of the control group were pink, while those in the model group showed a deeper red, increased mucus secretion, ulcers and swelling, and some capillary dilation.

[0118] According to the results of H&E staining, Figure 9 As shown in the figure, the mucosa of the blank group was normal in morphology, smooth and flat, without obvious differentiation, with tight muscle tissue, basically normal blood vessels and glands, and no obvious cell infiltration. In the model group, the mucosal epithelium was hyperplastic and thickened, and the integrity of the pharyngeal mucosal tissue was destroyed. There were more keratinized stratified cells, dilated and congested blood vessels, a small amount of glandular bleeding, loose muscle tissue, and inflammatory cell infiltration.

[0119] Routine blood counts in the rats showed that compared with the blank group, the model group had higher numbers of white blood cells, lymphocytes, and neutrophils, with a lower percentage of lymphocytes and a higher percentage of neutrophils. This suggests that the model group rats had inflammatory infection. Overall, the experimental model was successful.

[0120] Table 7 Routine blood parameters of rats

[0121]

[0122]

[0123] 2) In vivo toxicity experiments

[0124] 21) Weight and organ coefficient

[0125] Table 8 Organ coefficients

[0126]

[0127] Judging from the body weight and organ coefficients of each group, there was no significant change in body weight after administration, and there was no significant difference in organ coefficients between the administration group and the blank group. It was preliminarily judged that the drug was non-toxic or negligible to rats.

[0128] 22) Routine Blood Test and Biochemistry: Refer to the statistical charts for the routine blood test and biochemistry data for each animal experiment group. The routine blood test and biochemical function index data show that the model group showed a slight increase in serum white blood cell and lymphocyte counts compared to the blank group, with statistically significant differences. Each treatment group showed varying degrees of decrease compared to the model group, indicating that the model group had inflammation, while each treatment group had varying degrees of effect. There were no significant differences in the remaining indicators. Therefore, it is concluded that the microcapsules did not significantly damage blood, cardiac, liver, or kidney function.

[0129] 23) Internal organs HE staining: Refer to the HE staining images of the liver, lung, and kidney of rats in each group. No significant pathological changes were observed in the drug-treated groups or the blank control group, demonstrating that the drug has no significant organic toxicity to these organs.

[0130] 3) In vivo pharmacodynamics experiments

[0131] 31) Laryngoscopic Observation: Laryngoscopic results showed that compared with the model group, all treatment groups showed varying degrees of symptom relief, including lighter redness, decreased salivation, and relief of swelling and ulcers. The efficacy of the mixed group was significantly higher than that of the single-drug group, while the efficacy of the microcapsule group was higher than that of the free drug group. The efficacy of the microcapsules was also dose-dependent.

[0132] 32) Throat H&E staining: Pharyngeal mucosal integrity was improved in all drug-treated groups compared to the model group, with reduced inflammatory cell infiltration and more compact muscle tissue. The microcapsule group showed a significantly better effect than the free drug group. The pharyngeal mucosa was intact and smooth, with more compact muscle tissue. The efficacy of the microcapsule group increased in the low, medium, and high dose groups.

[0133] 33) Inflammatory Factor Detection: Referring to the serum inflammatory factor level chart, the model group showed significant increases in pro-inflammatory factors including tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6) compared to the blank group, indicating inflammation. Pro-inflammatory factors decreased to varying degrees in each treatment group compared to the model group. With the exception of the positive drug, the microcapsule group showed the greatest efficacy, indicating that the formulation promoted drug absorption and enhanced efficacy, and that the effectiveness of the microcapsules increased with increasing concentration.

[0134] In summary, chronic pharyngitis is a common chronic inflammatory disease, often characterized by congestion, swelling, vasodilation, and increased mucus secretion in the throat. Its development is closely linked to inflammatory mediators such as IL-1β, TNF-α, and IL-6, as well as inflammatory cells such as lymphocytes and leukocytes. In this experiment, 2.5% ammonia was used as a model-inducing agent. The results showed that rats exhibited congestion, swelling, increased mucus, and increased inflammatory cell infiltration in the pharynx. Significant increases in leukocyte, lymphocyte, IL-1β, TNF-α, and IL-6 levels, indicating successful chronic pharyngitis modeling.

[0135] Proinflammatory cytokines play a crucial role in the inflammatory response. TNF-α is one of the core mediators of the inflammatory response, exacerbating it. TNF-α can induce vasodilation and increase vascular permeability, facilitating the entry of immune cells into lesions and exacerbating the inflammatory response. IL-6 plays a crucial role in both acute and chronic inflammation. It promotes systemic inflammatory responses by stimulating the liver to secrete acute-phase proteins such as C-reactive protein (CRP). IL-1β is a potent proinflammatory cytokine that can activate both local and systemic inflammatory responses. By binding to its receptor, it initiates a series of cell signaling pathways, inducing inflammatory, acute-phase, and immune responses. It also stimulates other immune cells (such as T cells and B cells) to release more inflammatory cytokines, such as IL-6 and TNF-α, further intensifying the inflammatory response. Experimental results showed that compared with the control group, the model group had significantly elevated levels of IL-1β, TNF-α, and IL-6, indicating that these proinflammatory cytokines mediate the development of chronic pharyngitis. These inflammatory factors were suppressed in different microcapsule dosage groups, suggesting that the prepared microcapsules may inhibit the occurrence of chronic pharyngitis by reducing the release of these inflammatory factors. However, the specific mechanism still needs further study.

[0136] Currently, Western medicine's first-line treatment for chronic pharyngitis is antibiotics. While effective, they are difficult to use long-term. Essential oils, in addition to their anti-inflammatory properties, have a fragrant aroma that makes them easily accepted by patients when administered via spray. The aroma of essential oils can also help patients maintain a positive mental state, further facilitating treatment.

[0137] In summary, this study conducted qualitative and quantitative analysis of tea tree essential oil and successfully prepared tea tree essential oil-(+)-borneol ionic liquid microcapsules. The drug encapsulation efficiency and loading capacity were determined, and the microcapsules demonstrated stability under room temperature storage conditions. In vivo animal experiments demonstrated that the microcapsules had no significant toxicity to the blood, liver, and kidneys, and could inhibit the formation of inflammatory cells and the production of pro-inflammatory factors, demonstrating a strong anti-chronic pharyngitis effect.

[0138] Application Example 1-2

[0139] Tobacco application case 1

[0140] The prepared microcapsules and blank microcapsules were dispersed in an ethanol solution and then injected into a certain brand of cigarette tobacco at a ratio of 9.3 mg / cigarette. The cigarette samples were equilibrated under the conditions specified in GB / T 16447-2004 and then smoked according to the method specified in GB / T 19609-2004. The results are shown in Table 9.

[0141] Table 9 Sensory evaluation results of conventional cigarettes with added tea tree essential oil and (+)-borneol ionic liquid microcapsules

[0142]

[0143]

[0144] As can be seen from Table 9, the sensory quality of the cigarettes added with tea tree essential oil and (+)-borneol ionic liquid microcapsules is better than that of the cigarettes added with blank ionic liquid microcapsules. Specifically, the cigarettes added with tea tree essential oil and (+)-borneol ionic liquid microcapsules have significantly improved spicy and cool style characteristics, slightly more obvious herbal aroma, fruity aroma and woody aroma, and a slight green aroma. The consistency of the puffs is good, and they have good application prospects.

[0145] Tobacco application case 2

[0146] The tea tree essential oil, (+)-borneol ionic liquid microcapsules, and blank ionic liquid microcapsules prepared in the example were added to the hollow cooling section of heat-not-burn cigarettes at a concentration of 9.3 mg / cigarette. The sensory quality of the resulting cigarette cartridges was evaluated according to the sensory evaluation requirements of the Shanghai Institute of Heated Cigarettes. The results are shown in Table 10.

[0147] Table 10 Sensory evaluation results of heat-not-burn cigarettes with tea tree essential oil and (+)-borneol ionic liquid microcapsules

[0148]

[0149] As can be seen from Table 10, the heat-not-burn cigarettes with the addition of tea tree essential oil and (+)-borneol ionic liquid microcapsules have significantly higher throat-soothing effects, tea aroma, and cooling taste than the control group. Compared with the blank sample, the heat-not-burn cigarettes with the addition of tea tree essential oil and (+)-borneol ionic liquid microcapsules have significantly improved cooling and spicy characteristics, and the consistency between puffs is good, indicating good application prospects.

[0150] Utilization of key pharmaceutical ingredients in microcapsules:

[0151] Forty cigarettes of each type were taken and divided into two groups. Each Cambridge filter disc collected the TPM of mainstream smoke from 20 cigarettes. After smoking the cigarette, five empty puffs were taken to allow the mainstream smoke to settle freely for 30 seconds. The Cambridge filter disc was removed and placed in a stoppered conical flask. 20 mL of extraction solution (V 水 :V 乙醇 =1:1), ultrasonic extraction was performed at room temperature for 30 min, and then the volume was made up to 25 mL with the extraction solution, and the sample was filtered through a 0.45 μm organic phase microporous membrane as the test sample.

[0152] To further verify the utilization of key active ingredients in tea tree essential oil and (+)-borneol ionic liquid microcapsules in cigarettes, the transfer of microcapsules in the aerosol of conventional cigarettes and heat-not-burn cigarettes was investigated. Figure 19 .from Figure 19 It can be seen that 4-terpineol, γ-terpinene and 2-camphor alcohol, the main components of tea tree essential oil and (+)-borneol ionic liquid microcapsules, are newly added substances. Their transfer rates in the mainstream smoke of heat-not-burn cigarettes are 27.3%, 42.4% and 26.1%, respectively, and their transfer rates in the mainstream smoke of conventional cigarettes are 15.3%, 31.6% and 18.7%, respectively. This shows that the key pharmacological ingredients in the microcapsules can be effectively transferred to the cigarette aerosol, but the utilization rate of key pharmacological ingredients in heat-not-burn cigarettes is slightly higher. This is mainly because in conventional cigarettes, microcapsules are mainly added to tobacco, and the combustion temperature of conventional cigarettes is relatively high at 600-900℃, which may cause thermal decomposition or oxidation of some substances. In addition, the interception of tobacco and filter rods in conventional cigarettes further reduces the transfer rate of key pharmacological ingredients.

[0153] Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. An anti-chronic pharyngitis microcapsule, characterized in that: The anti-chronic pharyngitis microcapsule consists of a core material and a wall material. The wall material comprises gelatin and gum arabic. The core material comprises tea tree essential oil, (+)-borneol and ionic liquid. The ionic liquid is prepared by reacting geranic acid with choline bicarbonate.

2. The anti-chronic pharyngitis microcapsule according to claim 1, characterized in that The molar ratio of geranic acid to choline bicarbonate is 2:1, the mass ratio of tea tree essential oil, (+)-borneol and ionic liquid is 1:1:2, the mass ratio of core material to wall material is 1:2, and the mass ratio of gelatin to gum arabic is 1:

1.

3. A method for preparing the anti-chronic pharyngitis microcapsule according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Preparation of ionic liquid: Geranic acid and choline bicarbonate were dissolved in methanol respectively, the two solutions were mixed and stirred, methanol and water generated by the reaction were removed by rotary evaporation, and the ionic liquid was obtained by vacuum drying; (2) Preparation of microemulsion: Tween-80 was dissolved in anhydrous ethanol to prepare an emulsifier solution, and the solution was ultrasonicated at room temperature for 5 minutes and then vortexed for 2 minutes until the solution was clear and transparent, indicating complete dissolution; tea tree essential oil and (+)-borneol were dissolved in ionic liquid and ultrasonicated until there were no solid particles and the solution was clear and transparent, and then the emulsifier solution was added to form a mixed solution, which was fully mixed as the oil phase. The oil phase was added dropwise to the aqueous phase until the solution changed from clear to turbid, and the addition was stopped. The solution was stirred and ultrasonicated to obtain a microemulsion; (3) Preparation of microcapsules: Gum arabic and gelatin were dissolved in water and mixed to obtain a composite wall material solution, which was added to a microemulsion. The pH was adjusted to 4.0 with 1% glacial acetic acid and stirred to form an emulsion. The pH was then adjusted to 6.0 with NaOH. TG enzyme was added to fix the microcapsule structure. The microcapsule solution was obtained after stirring for 6 hours. The ethanol and unencapsulated tea tree essential oil and (+)-borneol were removed by rotary evaporation. The remaining solution was collected as a microcapsule solution and vacuum freeze-dried to obtain microcapsule powder.

4. The preparation method according to claim 3, characterized in that In step (1), the molar ratio of geranic acid to choline bicarbonate is 2:1, the stirring time is 1 day, the rotary evaporation temperature is 40° C. to remove methanol, the rotary evaporation temperature is 60° C. to remove water, and the drying time is 48 hours.

5. The preparation method according to claim 3, characterized in that In step (2), the mass ratio of tea tree essential oil to ionic liquid is 1:2, the mass ratio of emulsifier Tween-80 to co-emulsifier anhydrous ethanol is 3:1, and the mass ratio of tea tree essential oil to emulsifier is 3:

1.

6. The preparation method according to claim 3, characterized in that In step (3), the mass ratio of the core material to the wall material is 1:2, the mass ratio of gelatin to gum arabic is 1:1, and the complex coagulation pH is 4.

0.

7. Use of the anti-chronic pharyngitis microcapsule according to any one of claims 1-2 in the preparation of heat-not-burn tobacco products, characterized in that: The amount of the microcapsules added to the heat-not-burn tobacco products is 9.3-18.6 mg / stick.

8. The use according to claim 7, characterized in that In the heat-not-burn tobacco product, microcapsules are added to the hollow cooling section of the filter rod, and the hollow cooling section is made of polypropylene fiber.

9. Use of the anti-chronic pharyngitis microcapsule according to any one of claims 1 to 2 in the preparation of an anti-chronic pharyngitis drug.

10. The use according to claim 9, characterized in that The dosage form of the medicine is spray, lozenge or pill.

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