Liposomes of citronellol, process for their preparation and use thereof

Abstract

This invention relates to the field of tobacco processing technology, specifically to citronellol liposomes, their preparation methods, and applications. This invention provides a technical solution for preparing citronellol liposomes based on a solvent injection method, encapsulating citronellol as a core material within a phospholipid bilayer. This achieves liposome formation under mild conditions of 55-65℃, reducing the preparation time to within 30 minutes, and achieving a citronellol retention rate >95%. Simultaneously, optimizing the phospholipid-cholesterol ratio enhances the membrane material's capacity to encapsulate hydrophobic citronellol, resulting in a stable encapsulation rate ≥85%, significantly higher than the 60-70% of cyclodextrin encapsulation technology. This minimizes the waste of active ingredients. The prepared nanoliposomes can be added to tobacco, filters, heated cigarette flavorings, or e-cigarette e-liquids to achieve sustained release and protection of citronellol, prolonging aroma duration and enhancing the flavor of the tobacco.

Description

Technical Field

[0001] This invention relates to the field of tobacco processing technology, specifically to citronellol liposomes, their preparation methods, and applications. Background Technology

[0002] The aroma quality of tobacco products is a core element affecting consumer experience. To improve the texture of cigarettes, the industry commonly adds natural flavorings such as citronellol. While citronellol possesses an elegant rose aroma, its small molecular weight (156.27 g / mol) and low boiling point (244°C) make it highly susceptible to volatilization and loss during cigarette production, storage, and smoking. Furthermore, citronellol contains unsaturated double bonds, making it sensitive to light, heat, and oxygen; long-term storage can lead to oxidative degradation and aroma deterioration.

[0003] To address the stability issues of volatile flavorings, the tobacco industry has developed various encapsulation technologies. Cyclodextrin inclusion technology encapsulates flavoring molecules through hydrophobic cavities, but its encapsulation efficiency is typically below 70%, and the inclusion complex is prone to dissociation in humid environments. Microencapsulation technology uses gelatin, gum arabic, and other wall materials prepared through spray drying, but the process involves high temperatures, which can easily lead to the loss of heat-sensitive flavorings and poses a risk of organic solvent residue.

[0004] In recent years, liposomes have been widely used in the pharmaceutical field as nanodelivery systems. Their phospholipid bilayer structure is similar to that of cell membranes, exhibiting excellent biocompatibility and controllable release characteristics. Theoretically, liposomes can achieve physical isolation and protection by embedding citronellol into the phospholipid bilayer or encapsulating it in an inner aqueous phase. However, traditional thin-film dispersion methods for preparing liposomes require rotary evaporation to form a phospholipid film, a process that is high-temperature (typically >50°C) and time-consuming, resulting in significant loss of volatile components such as citronellol. Furthermore, existing liposome technologies are mostly designed for drug molecules, and there are no reports of systematic process optimization targeting the physicochemical properties of citronellol (low boiling point, high volatility, hydrophobicity). Therefore, developing a mild, simple, and industrially suitable method for preparing citronellol liposomes, while ensuring their stability and sustained-release effect in tobacco products, is a pressing technical bottleneck in this field. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide citronellol liposomes, their preparation method and applications. The present invention provides a method for preparing citronellol liposomes based on solvent injection, in which citronellol is encapsulated as a core material in a phospholipid bilayer. By optimizing key parameters such as the phospholipid-cholesterol ratio, ethanol injection rate, temperature and buffer conditions, the prepared nanoliposomes can be added to tobacco, filters, heated cigarette flavorings or e-cigarette e-liquids to achieve sustained release and protection of citronellol, prolong the aroma duration and enhance the flavor of the tobacco.

[0006] The present invention provides citronellol liposomes, which are composed of citronellol, soybean lecithin and cholesterol in a mass ratio of (0.35~1.4):(1.75~5.83):(0.35~1.17).

[0007] Compared with the prior art, the present invention uses citronellol, soybean lecithin and cholesterol as raw materials to prepare citronellol liposomes. The three are compatible with each other and have synergistic effects. The prepared citronellol liposomes have strong stability, high encapsulation rate and high sustained release function.

[0008] In some embodiments, the composition includes citronellol, soy lecithin, and cholesterol in a mass ratio of 1:(3.5~5.83):(0.7~1.17).

[0009] In some embodiments, the composition includes citronellol, soy lecithin, and cholesterol in a mass ratio of 1:3.5:0.7.

[0010] In some embodiments, the particle size of the citronellol liposomes is 100~300nm.

[0011] This invention provides a method for preparing the citronellol liposomes, characterized by comprising the following steps:

[0012] Step 1: Dissolve soybean lecithin, cholesterol, and emulsifier in a solvent to obtain the organic phase;

[0013] Step 2: Dissolve citronellol in the organic phase described in Step 1 to obtain a mixture;

[0014] Step 3: Take the mixture from Step 2 and mix it with the buffer solution. After stirring, removing the organic solvent, sonicating, filtering, and drying, citronellol liposomes are obtained.

[0015] In some embodiments, in step 1, the emulsifier includes at least one of Tween-80, poloxamer 188, and Span-80;

[0016] The mass ratio of soybean lecithin, cholesterol, and emulsifier is (4~8):1:(0.2~1).

[0017] In some specific embodiments, the emulsifier is Tween-80, and the mass ratio of soybean lecithin, cholesterol and Tween-80 is 3.5:0.7:0.35.

[0018] In some embodiments, in step 1, the solvent is ethanol;

[0019] The reaction temperature in step 1 is 50~60℃.

[0020] In some specific embodiments, in step 1, the solvent is ethanol, the reaction temperature in step 1 is 50°C, and the mass-to-volume ratio of soybean lecithin to ethanol is 3.5g:60mL.

[0021] In some embodiments, step 3, the mixing includes adding the mixture dropwise to a buffer solution under stirring conditions, wherein:

[0022] The dropping rate is 0.2~5 mL / min, the mixing reaction temperature is 55~65℃, and the stirring speed is 800~1200 rpm;

[0023] The buffer solution is PBS buffer, the concentration of the buffer solution is 5~30mM, and the pH of the buffer solution is 5.5~7.5;

[0024] The final concentration of ethanol in the mixture is 4.9 vol% to 14.6 vol.

[0025] In some specific embodiments, the dropping rate is 1 mL / min, the mixing reaction temperature is 60°C, and the stirring speed is 1000 rpm;

[0026] The buffer solution is PBS buffer, the concentration of the buffer solution is 10 mM, and the pH of the buffer solution is 6.8;

[0027] The final concentration of ethanol in the mixture is 7.1 vol.

[0028] In some embodiments, in step 3,

[0029] The stirring time is 10-30 minutes;

[0030] The removal of organic solvents is achieved by rotary evaporation and / or dialysis.

[0031] The ultrasonic power for ultrasonic homogenization is 200~400W, and the time is 2~5min;

[0032] The filtration process uses a filter membrane with a pore size of 0.22 μm.

[0033] In some specific embodiments, the stirring time is 20 minutes;

[0034] The rotary evaporation method specifically involves rotary evaporation at 45°C for 30 minutes.

[0035] The ultrasonic homogenization process uses an ultrasonic power of 300W and a duration of 3 minutes.

[0036] In some embodiments, step 3, the drying includes: mixing the filtered product with a freeze-drying protectant, pre-freezing, and freeze-drying to obtain citronellol liposomes;

[0037] The freeze-drying protectant includes mannitol or trehalose, and the mass ratio of the freeze-drying protectant to the soybean lecithin is (0.25~1):1;

[0038] The pre-freezing temperature is -50℃ to -40℃, and the time is 4 to 6 hours. The freeze-drying time is 36 to 48 hours.

[0039] In some specific embodiments, the freeze-drying protectant is mannitol, and the mass ratio of the freeze-drying protectant to the soybean lecithin is 0.5:1;

[0040] The pre-freezing temperature is -45℃ and the time is 5 hours, and the freeze-drying time is 48 hours.

[0041] This invention provides the application of the citronellol liposomes described above or the citronellol liposomes prepared by the method described above in the preparation of tobacco products.

[0042] The present invention provides tobacco products, including the citronellol liposomes described above or the citronellol liposomes prepared by the preparation method described above.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. This invention provides a technical solution for preparing citronellol liposomes based on a solvent injection method, encapsulating citronellol as a core material within a phospholipid bilayer. This achieves liposome formation under mild conditions of 55-65℃, reducing the preparation time to within 30 minutes, and maintaining a citronellol retention rate of >95%. Simultaneously, optimizing the phospholipid-cholesterol ratio enhances the membrane material's capacity to encapsulate hydrophobic citronellol, resulting in a stable encapsulation rate of ≥85%, significantly higher than the 60%-70% of cyclodextrin encapsulation technology, thus minimizing the waste of active ingredients.

[0045] 2. The liposomes prepared by this invention have a particle size of 100-300 nm, a PDI < 0.30, a Zeta potential of -20 to -30 mV, strong electrostatic repulsion, and no significant aggregation after 6 months of storage. Transmission electron microscopy shows that the vesicles are intact, and cholesterol embedded in the phospholipid bilayer enhances the membrane rigidity, reduces the photodegradation rate of citronellol by 70%, increases the thermal decomposition temperature by 15℃, and significantly improves the antioxidant capacity. When applied to tobacco products, the shelf life is extended by 2-3 times.

[0046] 3. This invention utilizes liposomes to physically isolate citronellol within a phospholipid bilayer, allowing it to be gradually released during cigarette combustion as the temperature rises. This results in an aroma retention time 2.5 times longer than direct addition. When smoked, the uniformity of the aroma across the initial, middle, and final stages is improved, smoke irritation is reduced by 30%, and the rose fragrance becomes more elegant and lingering. Only 0.01% to 0.5% is needed to achieve the desired effect, making it economical.

[0047] 4. This invention does not require expensive rotary evaporation film-forming equipment; it only requires conventional stirring, a constant flow pump (or syringe), and a temperature control device, resulting in low investment costs. The process cycle is short (approximately 1 hour), easily facilitating continuous operation. A 10L scale-up experiment showed batch-to-batch variation of <5%. Organic solvent residues can be easily controlled to <50 ppm through rotary evaporation or dialysis, meeting food-grade safety standards and possessing industrial production potential, making it suitable for widespread application. Attached Figure Description

[0048] Figure 1 Flowchart of solvent injection method preparation;

[0049] Figure 2 This is a SEM image of the liposome suspension in Example 1 of this invention;

[0050] Figure 3 The image shows a SEM image of the liposome suspension of Comparative Example 6 in this invention. Detailed Implementation

[0051] This invention provides citronellol liposomes, their preparation method, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0052] This invention employs a solvent injection method to prepare citronellol liposomes, encapsulating citronellol as a core material within a phospholipid bilayer. By optimizing key parameters such as the phospholipid-cholesterol ratio, ethanol injection rate, temperature, and buffer conditions, nanoliposomes with a particle size of 100-300 nm, encapsulation efficiency ≥85%, and polydispersity index (PDI) <0.30 are achieved. This preparation process eliminates the need for high-temperature rotary evaporation, significantly reducing the loss of volatile citronellol (retention rate >95%). The resulting liposomes can be added to tobacco, filters, heated cigarette flavorings, or e-cigarette e-liquids (addition amount 0.01-0.5%) to achieve sustained release and protection of citronellol, prolonging aroma duration and enhancing the flavor of tobacco.

[0053] Explanation of related terms

[0054] Citronellol: A monoterpenoid flavor compound with the molecular formula C 10 H 20 O, which has a rose-like aroma, is the active ingredient in the core material of the liposomes of this invention.

[0055] Liposomes are nanoscale vesicle structures composed of phospholipid bilayers that can encapsulate water-soluble or lipid-soluble substances, exhibiting good biocompatibility and excellent sustained-release properties.

[0056] Ethanol Injection Method: This method involves rapidly injecting an ethanol solution containing phospholipids and active ingredients into an aqueous phase, allowing phospholipid molecules to self-assemble into liposomes, without requiring a high-temperature evaporation step.

[0057] Encapsulation efficiency: The percentage of active ingredient encapsulated by liposomes out of the total added amount, calculated as (encapsulated amount / total amount) × 100%.

[0058] Polydispersity Index (PDI): An index that characterizes the uniformity of liposome particle size distribution. The smaller the value (<0.30), the more uniform and stable the system.

[0059] Zeta potential: the surface charge density of liposomes. In this invention, it is controlled at -20 to -30 mV to ensure the electrostatic repulsion stability of the system.

[0060] Phosphatidylcholine (PC): The main functional component of phospholipids. This invention requires that the PC content in soybean lecithin be ≥90%.

[0061] PBS (Phosphate Buffered Saline): Phosphate-buffered saline solution used to maintain pH and ionic strength stability during liposome formation.

[0062] Disadvantages of existing technology:

[0063] 1. Significant loss of active ingredients: The high-temperature rotary evaporation process of the thin-film dispersion method leads to severe volatilization loss of citronellol, resulting in low retention rate (<70%) and low raw material utilization; although cyclodextrin inclusion does not require high temperature, the encapsulation capacity is limited and the drug loading is low.

[0064] 2. Insufficient encapsulation efficiency: Existing methods generally achieve encapsulation rates of less than 80% for hydrophobic citronellol, leaving a large amount of free citronellol at risk of oxidation and volatilization.

[0065] 3. Complex processes or harsh conditions: Thin-film dispersion requires multiple steps and solvent residue is difficult to control; cyclodextrin inclusion requires precise control of temperature and concentration, resulting in poor process repeatability.

[0066] 4. Poor sustained-release performance: Cyclodextrin inclusion complexes are prone to dissociation in the humid environment of tobacco, and cannot achieve aroma retention; traditional liposomes have a wide particle size distribution (PDI>0.4), poor stability, and are prone to aggregation and sedimentation during storage.

[0067] 5. Difficult to industrialize: Thin film dispersion is difficult to produce continuously, and the scale-up effect is obvious; the cyclodextrin inclusion process generates a large amount of wastewater, resulting in high environmental costs.

[0068] In view of the shortcomings of the prior art, the present invention aims to provide citronellol liposomes and their preparation method, so as to achieve the following objectives:

[0069] 1. The solvent injection method replaces the traditional thin-film dispersion method, eliminating the need for high-temperature rotary evaporation in the preparation process, significantly reducing the loss of citronellol volatility, and achieving a retention rate of >95%;

[0070] 2. By optimizing the phospholipid-cholesterol ratio, ethanol injection rate, temperature, and buffer conditions, an encapsulation efficiency of ≥85% was achieved, with a particle size of 100-300 nm and a narrow distribution (PDI<0.30).

[0071] 3. Improves the light stability, thermal stability, and antioxidant properties of citronellol, extending the shelf life of tobacco products;

[0072] 4. Achieve sustained release of citronellol during cigarette combustion, extending the aroma duration by 2-3 times and reducing smoke irritation;

[0073] 5. Develop a preparation method that is simple, mild, requires no complex equipment, and is easy to scale up industrially, while controlling organic solvent residues to meet food-grade standards (<50 ppm).

[0074] This invention relates to a citronellol liposome and its solvent injection preparation process, the specific technical solution of which is as follows:

[0075] 1. Composition of liposome formulation

[0076] The liposomes are composed of a core material and a phospholipid bilayer membrane material, with the following specific mass ratio (based on the total mass of citronellol and phospholipids):

[0077] Citronellol (core material): 10-30 wt%

[0078] Phospholipids (main component of membrane material): 70-90 wt%

[0079] Cholesterol (membrane material conditioner): The mass ratio of cholesterol to phospholipids is 1:4-1:8.

[0080] Emulsifier (optional): Selected from Tween-80, poloxamer 188, or Span-80, with a mass ratio of 1:10 to 1:20 to phospholipids.

[0081] Freeze-drying protectant (optional): mannitol or trehalose, with a mass ratio of 0.25:1 to 1:1 to phospholipids.

[0082] Phospholipid requirements: Soy lecithin, egg yolk lecithin, hydrogenated soybean lecithin, or dipalmitoylphosphatidylcholine are preferred, or one or more combinations thereof; wherein the phosphatidylcholine content of soybean lecithin is ≥90% to ensure membrane flowability and encapsulation efficiency.

[0083] Liposome performance indicators:

[0084] Average particle size: 100-300 nm

[0085] Polydispersity Index (PDI): <0.30

[0086] Encapsulation rate: ≥85%

[0087] Zeta potential: -20 to -30 mV

[0088] Organic solvent residue: <50 ppm

[0089] 2. Solvent injection method for preparation

[0090] This invention uses a solvent injection method to prepare citronellol liposomes, eliminating the need for high-temperature rotary evaporation for film formation. The specific steps are as follows:

[0091] Step (1): Oil phase preparation

[0092] Dissolve phospholipids and cholesterol in anhydrous ethanol, add citronellol, and stir in a 50-60°C water bath until a clear solution is formed. Control the concentration of phospholipids in ethanol to be 40-80 mM, and the mass ratio of citronellol to phospholipids to be 1:4-1:8 (this ratio ensures that citronellol is fully dissolved in the phospholipid ethanol solution, laying the foundation for subsequent efficient encapsulation).

[0093] Step (2): Ethanol injection and liposome formation

[0094] At 55-65°C and a stirring speed of 800-1200 rpm, the ethanol solution from step (1) is slowly injected into the PBS buffer at a rate of 0.5-1.5 mL / min, controlling the final ethanol concentration to not exceed 7.5% v / v. The PBS buffer needs to be preheated to the same temperature, with a pH of 6.5-7.0 and an ionic strength of 10-20 mM. Slow injection ensures that phospholipid molecules self-assemble in an orderly manner to form monolayer vesicles, avoiding uneven particle size caused by excessively high local concentrations.

[0095] Step (3): Solvent removal

[0096] After injection, continue stirring for 10-30 minutes to stabilize the system. Then, remove residual ethanol by rotary evaporation (45°C, 30 minutes) or dialysis (MW 8000-14000, 4°C, 14 hours). Rotary evaporation is highly efficient and suitable for industrial production; dialysis is gentler and suitable for small-scale laboratory tests.

[0097] Step (4) (optional): Particle size homogenization treatment

[0098] The obtained liposome suspension was subjected to ultrasonic treatment (probe ultrasound, power 200-400 W, time 2-5 minutes) or high-pressure homogenization (pressure 800-1200 bar, cycle 3-5 times) to further reduce the particle size and improve the uniformity of distribution.

[0099] Step (5) (optional): Freeze-drying

[0100] Add mannitol or trehalose (to phospholipids by mass ratio of 0.25:1-1:1) as a freeze-drying protectant to the liposome suspension, pre-freeze at -50°C to -40°C for 4-6 hours, and then freeze-dry for 36-48 hours to obtain citronellol freeze-dried liposome powder. The freeze-dried product is easy to store and transport for a long time, and its performance is stable after reconstitution.

[0101] The following were explored in the preliminary research of this invention:

[0102] (1) Alternatives to preparation methods

[0103] Reverse evaporation method: Phospholipids and citronellol are dissolved in diethyl ether, and an aqueous phase is added and sonicated to form a W / O type emulsion. The organic solvent is removed by vacuum evaporation to form a gel, which is then dispersed with buffer solution. This method can achieve an encapsulation efficiency of 80-85%, but it uses a large amount of organic solvent, has a high risk of residue, and the process is complex, making it unsuitable for industrial application.

[0104] Microfluidic mixing: This method utilizes microfluidic chips to achieve precise and rapid mixing of ethanol and aqueous phases, allowing for accurate control of particle size (100-200 nm) and a PDI < 0.20. However, this equipment is expensive and currently limited to laboratory research; large-scale production still faces technical bottlenecks.

[0105] Supercritical fluid method: This method uses supercritical CO2 to dissolve phospholipids and citronellol, which then rapidly expand to form liposomes. This method leaves no organic solvent residue, but requires significant equipment investment, operates at high pressures (>100 bar), and has low technological maturity.

[0106] (2) Substitution of membrane material composition

[0107] Phospholipid substitution: Phosphatidylethanolamine (PE) and phosphatidylserine (PS) can be used to replace some of the phosphatidylcholine to adjust the charge and fluidity of the membrane material. However, PE is easily oxidized, and PS is expensive, and its overall performance is not as good as soybean lecithin.

[0108] Cholesterol substitution: Plant sterols such as stigmasterol and β-sitosterol can be used to replace cholesterol to meet the "cholesterol-free" claim. However, their effect on membrane rigidity adjustment is slightly inferior, and the encapsulation rate may decrease by 5-8%.

[0109] Emulsifier alternatives: Sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (CTAB) can be used to replace Tween-80. However, ionic emulsifiers may affect liposome stability and are subject to safety controversies, making them unsuitable for tobacco products.

[0110] (3) Alternatives to the buffer system

[0111] Citrate-sodium citrate buffer: pH 6.0-6.5, can replace PBS. However, its ionic strength is difficult to control and it may complex with phospholipids.

[0112] HEPES buffer: pH 7.0-7.5, better biocompatibility, but costs 5-10 times more than PBS, making it uneconomical for large-scale production.

[0113] (4) Improved version of thin film dispersion method

[0114] A thin-film dispersion method was employed, but after the phospholipid film was formed, a citronellol ethanol solution was added at a low temperature (4°C) for hydration to avoid high-temperature exposure. This method can achieve an encapsulation efficiency of 75-80%, but the operation is cumbersome, the hydration time is long (2-4 hours), and citronellol has low solubility at low temperatures, making it difficult to achieve high loading.

[0115] (5) Two-emulsion method

[0116] First, citronellol is dissolved in a small amount of ethanol, then added to an aqueous phospholipid solution and sonicated to form a primary emulsion. The primary emulsion is then added to the outer aqueous phase to form a W / O / W type double emulsion. Finally, the ethanol in the inner aqueous phase is evaporated to solidify the liposomes. Theoretically, this method can achieve an encapsulation efficiency of 90%, but the process is extremely difficult to control, with batch-to-batch variations exceeding 15%, resulting in low industrial feasibility.

[0117] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0118] Example 1

[0119] Preparation process flow as follows Figure 1 As shown, it specifically includes:

[0120] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0121] Add 1.0 g of citronellol and stir for 5 minutes until transparent to obtain an organic phase (i.e., an ethanol solution) containing citronellol.

[0122] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0123] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration of ethanol to be 7.1% v / v;

[0124] After the injection is complete, continue stirring for 20 minutes;

[0125] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0126] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0127] After passing through a 0.22 μm filter membrane, a liposome suspension was obtained.

[0128] Add mannitol (1.75 g, phospholipid:mannitol = 1:0.5), pre-freeze at –45°C for 5 hours, freeze-dry for 48 hours to obtain citronellol freeze-dried liposome powder;

[0129] Reconstitute with an equal volume of PBS before use.

[0130] Performance: Encapsulation efficiency 90.2±2.4%, particle size 180.87±1.47 nm, PDI 0.261±0.086, Zeta potential -20.32±5.31 mV, citronellol retention 95.3%.

[0131] Example 2

[0132] The liposome suspension prepared in Example 1 was uniformly sprayed onto the surface of tobacco at an addition rate of 0.08%. After equilibration at room temperature for 24 hours, it was rolled into standard cigarettes (cigarette weight 0.85±0.05 g). Smoking was simulated using a Borgwaldt RM20S smoking machine under ISO 3308 standard smoking conditions (35 mL / puff, 2 s / puff, 60 s interval). Each puff of smoke was individually captured through a Cambridge filter, and the citronellol content was determined by GC-MS (internal standard method, deuterated citronellol). The results are shown in Table 3 below. Seven trained professional sensory evaluators conducted a blind evaluation in a standard evaluation environment (22±1°C, 60±5% RH). Sensory scoring was based on a 9-point scale according to YC / T498-2014. The scoring criteria are shown in Table 1 below, and the scoring results are shown in Table 2 below.

[0133] Table 1. Sensory Evaluation Scoring Criteria (9-point scale)

[0134]

[0135] Table 2 Sensory rating results

[0136]

[0137] Table 3. Oral transfer rate of citronellol liposomes

[0138]

[0139] The coefficient of variation (CV) for each release is calculated as follows: (Standard deviation SD / Mean) × 100%

[0140] Liposome citronellol CV = 18.5%

[0141] The irritation of cigarette smoke was assessed using an in vitro cell model: Total particulate matter (TPM) was collected using a Borgwaldt LM5D smoking machine, and a cigarette smoke extract was prepared by ultrasonic extraction with physiological saline. This extract was then applied to human oral mucosal epithelial cells (HOECs). After 4 hours, cell viability and the levels of inflammatory factors IL-6 and IL-8 were measured. The irritation index was calculated using the formula, and the results are shown in Table 4.

[0142] Table 4 Stimulation Index

[0143]

[0144] Stimulation index = (1 − cell viability) × 0.5 + (IL-6 / 300) × 0.25 + (IL-8 / 350) × 0.25

[0145] The rate of reduction in irritation = (0.48 − 0.35) / 0.48 × 100% = 27.1%

[0146] Performance: Citronellol transfer rate in smoke is 18.7%, the main aroma lasts until the 6th puff, and there is still a slow release contribution at the end. The coefficient of variation for release per puff is 18.5%, the sensory score is improved by 1.2 points (out of 9), the smoke irritation is reduced by 27.1%, and the aroma is elegant and long-lasting.

[0147] Example 3

[0148] The process of Example 1 was scaled up to a 10L scale using an IKA KFS 25 constant flow pump (flow rate accuracy ±0.1 mL / min) and a Sartorius Biostat B temperature-controlled reactor (temperature control accuracy ±0.5°C), and three batches were produced continuously.

[0149] Weigh out 58.3 g of soybean lecithin (PC content ≥90%) and 11.7 g of cholesterol, dissolve them in 1 L of anhydrous ethanol, add 5.83 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0150] Add 16.7 g of citronellol and stir for 5 minutes until clear;

[0151] Preheat 11.7 L of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm;

[0152] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0153] After the injection is complete, continue stirring for 20 minutes;

[0154] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0155] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0156] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0157] Add mannitol (29.15 g), pre-freeze at -45°C for 5 hours, freeze-dry for 48 hours to obtain freeze-dried powder;

[0158] Reconstitute before use.

[0159] Performance: Average retention rate of 3 batches was 95.8±1.2%. Encapsulation efficiency was 89.7±1.5%, particle size was 182.5±2.8 nm, PDI was 0.274±0.032, Zeta potential was -19.8±4.1 mV, inter-batch variation was <5%, and ethanol residue was 42 ppm as detected by GC.

[0160] Comparative Example 1

[0161] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0162] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0163] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0164] Inject the ethanol solution at a rate of 0.2 mL / min, controlling the final concentration to 7.1% v / v;

[0165] After the injection is complete, continue stirring for 20 minutes;

[0166] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0167] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0168] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0169] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0170] Performance was measured after reconstitution with PBS before use.

[0171] Performance: Encapsulation efficiency 75.3±3.1%, particle size 245.3±5.2 nm, PDI 0.425±0.095, Zeta potential -18.5±4.2 mV, citronellol retention 82.1%.

[0172] Comparative Example 2

[0173] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0174] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0175] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0176] Inject the ethanol solution at a rate of 2.5 mL / min to control the final concentration at 7.1% v / v;

[0177] After the injection is complete, continue stirring for 20 minutes;

[0178] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0179] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0180] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0181] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0182] Performance was measured after reconstitution with PBS before use.

[0183] Performance: Encapsulation efficiency 71.2±4.5%, particle size 320.5±8.7 nm, PDI 0.512±0.128, Zeta potential -15.8±6.1 mV, citronellol retention 77.8%.

[0184] Comparative Example 3

[0185] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0186] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0187] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0188] Inject the ethanol solution at a rate of 5.0 mL / min, controlling the final concentration to 7.1% v / v;

[0189] After the injection is complete, continue stirring for 20 minutes;

[0190] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0191] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0192] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0193] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0194] Performance was measured after reconstitution with PBS before use.

[0195] Performance: Encapsulation efficiency 58.7±6.2%, particle size 458.7±15.3 nm, PDI 0.687±0.205, Zeta potential -8.2±5.4 mV, citronellol retention 54.2%.

[0196] Comparative Example 4

[0197] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0198] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0199] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 45°C and stir at 1000 rpm;

[0200] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0201] After the injection is complete, continue stirring for 20 minutes;

[0202] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0203] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0204] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0205] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0206] Performance was measured after reconstitution with PBS before use.

[0207] Performance: Encapsulation efficiency 68.4±2.8%, particle size 195.4±3.6 nm, PDI 0.356±0.087, Zeta potential -17.2±3.9 mV, citronellol retention 73.5%.

[0208] Comparative Example 5

[0209] Weigh 1.75 g of soybean lecithin (PC content ≥90%) and 0.35 g of cholesterol, dissolve them in 120 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0210] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0211] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0212] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 14.6% v / v;

[0213] After the injection is complete, continue stirring for 20 minutes;

[0214] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0215] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0216] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0217] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0218] Performance was measured after reconstitution with PBS before use.

[0219] Performance: Encapsulation efficiency 62.3±3.5%, particle size 165.2±4.1 nm, PDI 0.382±0.095, Zeta potential -14.5±4.7 mV, citronellol retention 68.7%.

[0220] Comparative Example 6

[0221] Weigh out 5.83 g of soybean lecithin (PC content ≥90%) and 1.17 g of cholesterol, dissolve them in 36 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0222] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0223] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0224] Inject the ethanol solution at a rate of 1.0 mL / min to control the final concentration at 4.9% v / v;

[0225] After the injection is complete, continue stirring for 20 minutes;

[0226] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0227] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0228] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0229] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0230] Performance was measured after reconstitution with PBS before use.

[0231] Performance: Encapsulation efficiency 79.5±2.1%, particle size 385.6±12.4 nm, PDI 0.498±0.112, Zeta potential -22.1±3.2 mV, citronellol retention 88.3%.

[0232] Comparative Example 7

[0233] Weigh 3.5 g of soybean lecithin (PC content ≥90%), dissolve it in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0234] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0235] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0236] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0237] After the injection is complete, continue stirring for 20 minutes;

[0238] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0239] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0240] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0241] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0242] Performance was measured after reconstitution with PBS before use.

[0243] Performance: Encapsulation efficiency 79.5±3.2%, particle size 165.8±3.9 nm, PDI 0.412±0.089, Zeta potential -12.4±5.1 mV, citronellol retention 90.2%.

[0244] Comparative Example 8

[0245] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.35 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0246] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0247] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0248] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0249] After the injection is complete, continue stirring for 20 minutes;

[0250] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0251] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0252] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0253] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0254] Performance was measured after reconstitution with PBS before use.

[0255] Performance: Encapsulation efficiency 79.8±2.5%, particle size 168.5±2.8 nm, PDI 0.298±0.076, Zeta potential -16.8±4.3 mV, citronellol retention 88.5%.

[0256] Comparative Example 9

[0257] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 1.75 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0258] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0259] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0260] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0261] After the injection is complete, continue stirring for 20 minutes;

[0262] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0263] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0264] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0265] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0266] Performance was measured after reconstitution with PBS before use.

[0267] Performance: Encapsulation efficiency 83.1±1.8%, particle size 415.2±8.7 nm, PDI 0.421±0.098, Zeta potential -28.5±2.1 mV, citronellol retention 91.2%.

[0268] The results above show that Comparative Example 7 had a higher retention rate than Comparative Example 8 because the low concentration of cholesterol increased the fluidity of the liposome membrane, making citronellol more prone to leakage. In contrast, Comparative Example 9, with its significantly increased cholesterol content (50%), resulted in a highly compacted lipid bilayer, significantly inhibiting the transmembrane diffusion of citronellol and thus improving its retention rate. Combining Comparative Examples 7-9, it can be concluded that cholesterol has a concentration-dependent bidirectional regulatory effect on liposome membrane permeability: low concentrations increase flow and promote release, while high concentrations result in denser and more stable membranes.

[0269] Comparative Example 10

[0270] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0271] Add 1.4 g of citronellol and stir for 5 minutes until clear;

[0272] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0273] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0274] After the injection is complete, continue stirring for 20 minutes;

[0275] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0276] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0277] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0278] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0279] Performance was measured after reconstitution with PBS before use.

[0280] Performance: Encapsulation efficiency 77.2±3.6%, particle size 340.5±9.8 nm, PDI 0.512±0.125, Zeta potential -18.9±5.2 mV, citronellol retention 85.3%.

[0281] Comparative Example 11

[0282] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0283] Add 0.35 g of citronellol and stir for 5 minutes until clear;

[0284] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0285] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0286] After the injection is complete, continue stirring for 20 minutes;

[0287] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0288] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0289] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0290] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0291] Performance was measured after reconstitution with PBS before use.

[0292] Performance: Encapsulation efficiency 68.4±4.1%, particle size 172.1±3.5 nm, PDI 0.265±0.067, Zeta potential -19.6±3.8 mV, citronellol retention 93.5%.

[0293] Comparative Example 12

[0294] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0295] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0296] Preheat 700 mL of PBS buffer (10 mM, pH 5.5) to 60°C and stir at 1000 rpm.

[0297] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0298] After the injection is complete, continue stirring for 20 minutes;

[0299] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0300] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0301] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0302] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0303] Performance was measured after reconstitution with PBS before use.

[0304] Performance: Encapsulation efficiency 74.6±3.2%, particle size 192.3±4.7 nm, PDI 0.341±0.087, Zeta potential -12.4±4.1 mV, citronellol retention 87.1%.

[0305] Comparative Example 13

[0306] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0307] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0308] Preheat 700 mL of PBS buffer (10 mM, pH 7.5) to 60°C and stir at 1000 rpm.

[0309] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0310] After the injection is complete, continue stirring for 20 minutes;

[0311] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0312] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0313] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0314] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0315] Performance was measured after reconstitution with PBS before use.

[0316] Performance: Encapsulation efficiency 76.8±2.9%, particle size 205.6±5.3 nm, PDI 0.356±0.092, Zeta potential -25.8±3.4 mV, citronellol retention 85.6%.

[0317] Comparative Example 14

[0318] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0319] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0320] Preheat 700 mL of PBS buffer (5 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0321] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0322] After the injection is complete, continue stirring for 20 minutes;

[0323] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0324] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0325] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0326] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0327] Performance was measured after reconstitution with PBS before use.

[0328] Performance: Encapsulation efficiency 72.5±3.8%, particle size 188.7±4.2 nm, PDI 0.382±0.095, Zeta potential -16.7±4.8 mV, citronellol retention 83.4%.

[0329] Comparative Example 15

[0330] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0331] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0332] Preheat 700 mL of PBS buffer (30 mM, pH 6.8) to 60°C and stir at 1000 rpm;

[0333] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0334] After the injection is complete, continue stirring for 20 minutes;

[0335] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0336] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0337] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0338] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0339] Performance was measured after reconstitution with PBS before use.

[0340] Performance: Encapsulation efficiency 69.8±4.5%, particle size 295.6±12.1 nm, PDI 0.465±0.128, Zeta potential -19.3±5.6 mV, citronellol retention 81.7%.

[0341] Comparative Example 16

[0342] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0343] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0344] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0345] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0346] After the injection is complete, continue stirring for 20 minutes;

[0347] Ethanol was removed by rotary evaporation at 45°C for 30 minutes; ultrasonic homogenization was omitted.

[0348] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0349] Add mannitol (1.75 g), pre-freeze at –45°C for 5 hours, and freeze-dry for 48 hours;

[0350] Performance was measured after reconstitution with PBS before use.

[0351] Performance: Encapsulation efficiency 88.7±2.1%, particle size 285.6±35.4 nm, PDI 0.425±0.156, Zeta potential -19.8±4.7 mV, citronellol retention 94.5%.

[0352] Comparative Example 17

[0353] Weigh out 3.5 g of soybean lecithin (PC content ≥90%) and 0.7 g of cholesterol, dissolve them in 60 mL of anhydrous ethanol, add 0.35 g of Tween-80, and stir to dissolve in a 50°C water bath.

[0354] Add 1.0 g of citronellol and stir for 5 minutes until clear;

[0355] Preheat 700 mL of PBS buffer (10 mM, pH 6.8) to 60°C and stir at 1000 rpm.

[0356] Inject the ethanol solution at a rate of 1.0 mL / min, controlling the final concentration to 7.1% v / v;

[0357] After the injection is complete, continue stirring for 20 minutes;

[0358] Remove ethanol by rotary evaporation at 45°C for 30 minutes;

[0359] Particle size homogenization was performed using ultrasound (300 W, 3 min) with a probe.

[0360] The liposome suspension was obtained by passing the solution through a 0.22 μm filter membrane.

[0361] Remove the freeze-drying protectant and proceed with freeze-drying directly (-50℃, 48h).

[0362] Performance: After lyophilization and reconstitution, the encapsulation efficiency decreased to 68.4%, the particle size increased from 185.3 nm to 382.5 nm, the PDI was 0.621±0.189, the Zeta potential was -12.4±6.3 mV, and the citronellol retention decreased to 72.3±1.8%.

[0363] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Citronellol liposomes, characterized in that, Its composition includes citronellol, soy lecithin and cholesterol in a mass ratio of (0.35~1.4):(1.75~5.83):(0.35~1.17).

2. The liposomes according to claim 1, characterized in that, Its composition includes citronellol, soy lecithin and cholesterol in a mass ratio of 1:(3.5~5.83):(0.7~1.17).

3. The liposomes according to claim 1 or 2, characterized in that, Its composition includes citronellol, soy lecithin, and cholesterol in a mass ratio of 1:3.5:0.

7.

4. The liposomes according to any one of claims 1 to 3, characterized in that, The particle size of the citronellol liposomes is 100~300nm.

5. The method for preparing citronellol liposomes according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: Dissolve soybean lecithin, cholesterol, and emulsifier in a solvent to obtain the organic phase; Step 2: Dissolve citronellol in the organic phase described in Step 1 to obtain a mixture; Step 3: Take the mixture from Step 2 and mix it with the buffer solution. After stirring, removing the organic solvent, sonicating, filtering, and drying, citronellol liposomes are obtained.

6. The preparation method according to claim 5, characterized in that, In step 1, the emulsifier includes at least one of Tween-80, poloxamer 188, and Span-80; The mass ratio of soybean lecithin, cholesterol, and emulsifier is (4~8):1:(0.2~1).

7. The preparation method according to claim 5, characterized in that, In step 1, the solvent is ethanol; The reaction temperature in step 1 is 50~60℃.

8. The preparation method according to claim 5, characterized in that, In step 3, the mixing includes adding the mixture dropwise to a buffer solution under stirring conditions, wherein: The dropping rate is 0.2~5 mL / min, the mixing reaction temperature is 55~65℃, and the stirring speed is 800~1200 rpm; The buffer solution is PBS buffer, the concentration of the buffer solution is 5~30mM, and the pH of the buffer solution is 5.5~7.5; The final concentration of ethanol in the mixture is 4.9 vol% to 14.6 vol.

9. The preparation method according to claim 5, characterized in that, In step 3 The stirring time is 10-30 minutes; The removal of organic solvents is achieved by rotary evaporation and / or dialysis. The ultrasonic power for ultrasonic homogenization is 200~400W, and the time is 2~5min; The filtration process uses a filter membrane with a pore size of 0.22 μm.

10. The preparation method according to claim 5, characterized in that, In step 3, the drying process includes: mixing the filtered product with a freeze-drying protectant, and then pre-freezing and freeze-drying to obtain citronellol liposomes; The freeze-drying protectant includes mannitol or trehalose, and the mass ratio of the freeze-drying protectant to the soybean lecithin is (0.25~1):1; The pre-freezing temperature is -50℃ to -40℃, and the time is 4 to 6 hours. The freeze-drying time is 36 to 48 hours.

11. The use of citronellol liposomes according to any one of claims 1 to 4 or citronellol liposomes prepared by the preparation method according to any one of claims 5 to 10 in the preparation of tobacco products.

12. A tobacco product, characterized in that, Includes citronellol liposomes as described in any one of claims 1 to 4 or citronellol liposomes prepared by the preparation method described in any one of claims 5 to 10.

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