Low-irritation nicotine gel as well as preparation method and application thereof

Abstract

The invention provides low-irritation nicotine gel as well as a preparation method and application thereof. The nicotine gel comprises a gel matrix and an exosome compound embedded in the gel matrix, the gel matrix is prepared from the following components: pullulan and water; the exosome compound comprises a pomegranate exosome and nicotine loaded in the pomegranate exosome. According to the pomegranate exosome, the nicotine is coated in the pomegranate exosome, so that the stability of the nicotine can be effectively improved, the skin permeability of the nicotine is enhanced, and the abstinence symptom occurring during smoking cessation is relieved; pulullan is used as a medium, the skin absorption effect of nicotine is further regulated and controlled, and the situation that the smoking cessation effect is affected by too fast or too slow absorption is avoided.

Description

Technical Field

[0001] This invention belongs to the field of topical formulation technology, specifically relating to a low-irritant nicotine gel, its preparation method, and its application. Background Technology

[0002] The main principle of nicotine replacement therapy is to provide some of the nicotine that would normally be obtained from cigarettes in a non-cigarette form. The therapeutic dose of nicotine is far lower than the amount obtained from cigarettes, which can reduce withdrawal symptoms and increase the success rate of quitting smoking, while avoiding the toxicity of harmful substances such as carbon monoxide and tar to the body.

[0003] CN112220756A discloses a nicotine granule composition and its preparation method. The composition mainly consists of immediate-release nicotine granules and sustained-release nicotine granules. The immediate-release nicotine granules primarily include: nicotine raw materials, fillers, binders, buffers, sweeteners, and flavorings. The sustained-release nicotine granules primarily include: nicotine raw materials, polymeric sustained-release materials, binders, buffers, sweeteners, and flavorings. The immediate-release and sustained-release nicotine granules ensure the speed at which the human body experiences pleasure and maintain a therapeutically effective concentration of nicotine in the body for a prolonged period.

[0004] CN104940408A discloses a hydrogel emergency smoking cessation patch formulation and a method for preparing the patch. The formulation comprises 0.1-2.0% natural tobacco extract, 1-15% nicotine inclusion complex, 0.5-3.0% peppermint extract, 5.0-10.0% sodium polyacrylate, 0.5-3.0% sodium carboxymethyl cellulose, 0.1-2.0% aluminum hydroxyl, 0.2-1.0% tartaric acid, 4.0-8.0% ethanol, 20.0-30.0% glycerin, and 45.0-60.0% purified water, which are then coated onto a substrate to obtain the smoking cessation patch. This patch features low nicotine dosage, good uniform and sustained-release effect, controllable safety, strong skin affinity, good adhesion, convenient portability and use, simple preparation process, and no secondary environmental pollution.

[0005] However, the effectiveness of existing smoking cessation products remains limited, especially patch-type products which have poor skin affinity and are highly irritating. Therefore, there is an urgent need to develop a low-irritant nicotine gel with better smoking cessation effects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a low-irritant nicotine gel, its preparation method, and its application. The nicotine gel of the present invention has a stable nicotine skin penetration rate and low irritation, effectively alleviating withdrawal symptoms during smoking cessation and reducing nicotine dependence.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a low-irritant nicotine gel, the nicotine gel comprising a gel matrix and an exosome complex embedded in the gel matrix;

[0009] The components of the gel matrix include pullulan and water;

[0010] The exosome complex includes pomegranate exosomes and nicotine loaded in pomegranate exosomes.

[0011] This invention encapsulates nicotine in pomegranate exosomes, which can effectively improve the stability of nicotine, enhance the skin permeability of nicotine, and reduce withdrawal symptoms during smoking cessation; pullulan polysaccharide, as a medium, further regulates the skin absorption of nicotine, avoiding absorption that is too fast or too slow from affecting the smoking cessation effect.

[0012] The phospholipid bilayer of pomegranate exosomes can fuse with skin cells, enabling nicotine penetration while delivering active ingredients such as proteins and RNA carried within to the deeper layers of the skin. This helps alleviate the irritation caused by nicotine and indirectly improves skin permeability by influencing the skin microenvironment, thereby regulating the amount of nicotine transdermally absorbed.

[0013] Preferably, the mass ratio of the gel matrix to the exosome complex is (10-15):(1-3).

[0014] The specific point values ​​in (10-15) can be 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15, etc.

[0015] The specific point values ​​in (1-3) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0016] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0017] Preferably, the components of the gel matrix include 1-10 parts pullulan and 70-85 parts water by weight.

[0018] The pullulan polysaccharide can be present in parts by weight of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0019] The weight percentage of the water can be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, or 85 parts, etc.

[0020] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0021] Preferably, the ratio of pomegranate exosomes to nicotine is (1-10)×10⁻⁶. 8 Quantity: (0.01-1) mg.

[0022] The specific point values ​​in (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0023] The specific point values ​​in (0.01-1) can be 0.01, 0.02, 0.05, 0.07, 0.1, 0.3, 0.5, 0.8 or 1, etc.

[0024] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0025] Preferably, the pomegranate exosomes are prepared by a method comprising the following steps:

[0026] Pomegranate fruit was mixed with buffer solution, crushed, and then mixed with coenzyme for enzymatic hydrolysis. The supernatant was collected by differential centrifugation and purified to obtain the pomegranate exosomes.

[0027] This invention utilizes enzyme-assisted extraction of pomegranate exosomes, which can effectively dissolve impurities and enhance exosome activity.

[0028] Preferably, the ratio of pomegranate fruit to buffer solution is 1 g:(1.5-3) mL.

[0029] The specific point values ​​in (1.5-3) can be 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.5, 2.8 or 3, etc.

[0030] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0031] Preferably, the buffer solution includes any one or a combination of at least two of PBS buffer, HEPES buffer, or Tris-HCl buffer.

[0032] Preferably, the mass ratio of pomegranate fruit to coenzyme is 1:(0.001-0.01).

[0033] The specific point values ​​in (0.001-0.01) can be 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 or 0.01, etc.

[0034] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0035] Preferably, the coenzyme includes any one or a combination of at least two of polygalacturonase, arabinosidase, or β-glucosidase.

[0036] Preferably, the coenzyme includes polygalacturonase, arabinosidase, and β-glucosidase.

[0037] A combination of polygalacturonase, arabinosidase, and β-glucosidase was selected for the extraction of pomegranate exosomes. The three enzymes have a synergistic effect, dissolve interfering impurities, ensure the integrity and activity of pomegranate exosomes, have a better regulatory effect on nicotine transdermal delivery, and significantly reduce irritation.

[0038] Preferably, the mass ratio of polygalacturonase, arabinosidase and β-glucosidase is (1-3):(1-5):(1-8).

[0039] The specific point values ​​in (1-3) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0040] The specific point values ​​in (1-5) can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0041] The specific point values ​​in (1-8) can be 1, 2, 3, 4, 5, 6, 7 or 8, etc.

[0042] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0043] Preferably, the temperature of the enzymatic hydrolysis reaction is 30-40℃, for example, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, etc.; the time is 1-3 h, for example, 1 h, 1.1 h, 1.2 h, 1.5 h, 1.7 h, 2 h, 2.3 h, 2.5 h, 2.8 h or 3 h, etc.

[0044] Preferably, the differential centrifugation includes three centrifugations: the first centrifugation is performed at a speed of 2000-4000 g, for example, 2000 g, 2200 g, 2500 g, 2700 g, 3000 g, 3300 g, 3500 g, 3800 g, or 4000 g, etc.; the time is 5-15 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min, etc.; the second centrifugation is performed at a speed of 7000-9000 g, for example, 7000 g, 7200 g, 7500 g, 7700 g, 8000 g, 8300 g, 8500 g, 8800 g, or 9000 g, etc.; the time is 15-25 min, for example, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, etc. The centrifugation times are 22 min, 23 min, 24 min, or 25 min, etc.; the third centrifugation speed is 9000-11000 g, for example, 9000 g, 9200 g, 9500 g, 9700 g, 10000 g, 10300 g, 10500 g, 10800 g, or 11000 g, etc.; the time is 55-65 min, for example, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, or 65 min, etc.

[0045] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0046] Preferably, the purification method includes tangential flow filtration.

[0047] Tangential flow filtration improves the purity of exosomes while removing enzymes.

[0048] Preferably, the pullulan is prepared by a method comprising the following steps:

[0049] Citrus, extractant, and water are mixed and subjected to enzymatic hydrolysis to inactivate the enzyme and obtain citrus hydrolysate. *Bacillus buddingus* is inoculated into a culture medium containing the citrus hydrolysate and fermented. After centrifugation, the supernatant is collected, mixed with ethanol, and the precipitate is collected to obtain pullulan polysaccharide.

[0050] Adding citrus hydrolysate during the fermentation of *Brachystomata buddingus* can improve the molecular weight and glycosidic bond ratio of pullulan, affecting the three-dimensional network structure of the gel matrix formed by pullulan, thus having a better regulatory effect on nicotine transdermal delivery and significantly reducing irritation.

[0051] Preferably, the mass ratio of the citrus fruit to the extracting enzyme is 1:(0.01-0.1).

[0052] The specific point values ​​in (0.01-0.1) can be 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1, etc.

[0053] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0054] Preferably, the extraction enzyme includes any one or a combination of at least two of protease, laccase, or fruit acid enzyme.

[0055] Preferably, the extraction enzymes include protease, laccase, and fruit acid enzyme.

[0056] Citrus hydrolysate was prepared by using a combination of protease, laccase, and fruit acid enzyme. The three enzymes have a synergistic effect. The citrus hydrolysate prepared by the combination affects the fermentation and metabolism of *Brachystomata buddingus*, thereby affecting the structure of pullulan polysaccharide gel matrix, and has a better regulatory effect on nicotine transdermal absorption, significantly reducing irritation.

[0057] Preferably, the mass ratio of the protease, laccase, and fruit acid enzyme is (1-8):(1-5):(1-5).

[0058] The specific point values ​​in (1-8) can be 1, 2, 3, 4, 5, 6, 7 or 8, etc.

[0059] The specific point values ​​in the first (1-5) can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0060] The specific point values ​​in the second (1-5) can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0061] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0062] Preferably, the temperature of the enzymatic hydrolysis reaction is 35-45℃, for example, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃, etc.; and the time is 20-40 min, for example, 20 min, 22 min, 25 min, 27 min, 30 min, 33 min, 35 min, 38 min or 40 min, etc.

[0063] Preferably, the enzyme inactivation temperature is 110-130℃, for example, 110℃, 112℃, 115℃, 117℃, 120℃, 123℃, 125℃, 128℃ or 130℃, etc.; the time is 5-10 min, for example, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min or 10 min, etc.

[0064] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0065] Preferably, the enzyme inactivation process further includes a concentration and drying step.

[0066] Preferably, the viable count of the budding short-stem mold at the start of the fermentation system is (1-5)×10⁻⁶. 9 CFU / mL.

[0067] The specific point values ​​in (1-5) can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0068] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0069] Preferably, the citrus enzymatic hydrolysate has a mass percentage of 5-15 wt% in the culture medium, for example, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, or 15 wt%.

[0070] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0071] Preferably, the culture medium includes a carbon source, a nitrogen source, inorganic salts, and water.

[0072] Preferably, the fermentation temperature is 27-35℃, for example, 27℃, 27.5℃, 28℃, 28.5℃, 29℃, 29.5℃, 30℃, 31℃, 32℃, 33℃, 34℃, or 35℃; and the time is 20-30 h, for example, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, or 30 h.

[0073] Preferably, the centrifugation speed is 10000-12000 rpm, for example, 10000 rpm, 10200 rpm, 10500 rpm, 10700 rpm, 11000 rpm, 11300 rpm, 11500 rpm, 11800 rpm, or 12000 rpm; the time is 10-20 min, for example, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min.

[0074] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0075] Preferably, the volume ratio of the supernatant to ethanol is 1:(1.5-3).

[0076] The specific point values ​​in (1.5-3) can be 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.5, 2.8 or 3, etc.

[0077] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0078] Preferably, the collection of sediment further includes a washing and / or drying step.

[0079] Preferably, the components of the gel matrix further include a cooling agent.

[0080] Preferably, the components of the gel matrix further include 0.1-2 parts by weight of a cooling agent.

[0081] The cooling agent can be present in parts by weight of 0.1, 0.2, 0.5, 0.7, 1, 1.3, 1.5, 1.8, or 2 parts, etc.

[0082] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0083] Preferably, the cooling agent comprises any one or a combination of at least two of menthol, peppermint oil, menthol, menthone, ethyl menthane formamide, menthyl acetate, or limonene.

[0084] Preferably, the combination of the gel matrix further includes a moisturizer.

[0085] Preferably, the components of the gel matrix further include 1-5 parts by weight of a humectant.

[0086] The humectant can be present in parts by weight of 1 part, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, or 5 parts, etc.

[0087] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0088] Preferably, the moisturizer comprises any one or a combination of at least two of the following: ceramide, β-sitosterol, hyaluronic acid, trehalose, hydroxyethyl urea, asiaticoside, panthenol, or allantoin.

[0089] In a second aspect, the present invention provides a method for preparing nicotine gel as described in the first aspect, the method comprising:

[0090] Pomegranate exosomes and nicotine were mixed and sonicated to obtain the exosome complex; pullulan was mixed with water to prepare a gel matrix; the gel matrix and the exosome complex were mixed to obtain the nicotine gel.

[0091] Preferably, the preparation of the gel matrix further includes the addition of a cooling agent and / or a moisturizing agent.

[0092] Preferably, the power of the ultrasound is 100-300 W, for example, 100 W, 120 W, 150 W, 170 W, 200 W, 230 W, 250 W, 280 W or 300 W; the duration is 15-30 min, for example, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 22 min, 25 min, 28 min or 30 min.

[0093] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0094] Thirdly, the present invention provides the application of the nicotine gel as described in the first aspect in reducing nicotine dependence, wherein the nicotine gel is used by means of applying it to the skin.

[0095] Compared with the prior art, the present invention has the following beneficial effects:

[0096] This invention encapsulates nicotine in pomegranate exosomes, which can effectively improve the stability of nicotine, enhance the skin permeability of nicotine, and reduce withdrawal symptoms during smoking cessation; pullulan polysaccharide, as a medium, further regulates the skin absorption of nicotine, avoiding absorption that is too fast or too slow from affecting the smoking cessation effect. Detailed Implementation

[0097] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0098] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0099] The sources of materials used in the following specific embodiments are as follows:

[0100] The budding short-stemmed mold was obtained from the China General Microbiological Culture Collection Center, accession number CGMCC NO.13178.

[0101] Prepare the basal culture medium: Dissolve 3 wt% glucose, 1 wt% yeast extract, 2 wt% peptone, 0.5 wt% dipotassium hydrogen phosphate, 0.05 wt% magnesium sulfate, 0.03 wt% ammonium sulfate and 0.05 wt% sodium chloride in water, and sterilize at 121℃ for 15 min.

[0102] Preparation Example 1

[0103] This preparation example provides a pomegranate exosome, which is prepared by a method including the following steps:

[0104] 100 g of pomegranate fruit was added to 200 mL of PBS buffer and thoroughly crushed. Then, 0.2 g of polygalacturonase, 0.2 g of arabinoside enzyme, and 0.1 g of β-glucosidase were added and mixed thoroughly. The mixture was reacted at 35 °C for 2 h to obtain the enzymatic hydrolysate. The hydrolysate was first centrifuged at 3000 g for 10 min and the supernatant was collected; then centrifuged at 8000 g for 20 min and the supernatant was collected; finally, it was centrifuged at 10000 g for 60 min and the supernatant was collected; the mixture was then filtered tangentially to obtain the pomegranate exosomes.

[0105] Preparation Example 2

[0106] This preparation example provides a pomegranate exosome, which is prepared by a method including the following steps:

[0107] 100 g of pomegranate fruit was added to 300 mL of PBS buffer and thoroughly crushed. Then, 0.3 g of polygalacturonase, 0.5 g of arabinoside enzyme, and 0.2 g of β-glucosidase were added and mixed thoroughly. The mixture was reacted at 30 °C for 3 h to obtain the enzymatic hydrolysate. The hydrolysate was first centrifuged at 4000 g for 5 min and the supernatant was collected; then centrifuged at 7000 g for 25 min and the supernatant was collected; finally, it was centrifuged at 11000 g for 55 min and the supernatant was collected; the mixture was then filtered tangentially to obtain the pomegranate exosomes.

[0108] Preparation Example 3

[0109] This preparation example provides a pomegranate exosome, which is prepared by a method including the following steps:

[0110] 100 g of pomegranate fruit was added to 150 mL of PBS buffer and thoroughly crushed. Then, 0.1 g of polygalacturonase, 0.1 g of arabinoside, and 0.1 g of β-glucosidase were added and mixed thoroughly. The mixture was reacted at 40 °C for 1 h to obtain the enzymatic hydrolysate. The hydrolysate was first centrifuged at 2000 g for 15 min and the supernatant was collected; then centrifuged at 9000 g for 15 min and the supernatant was collected; then centrifuged at 9000 g for 65 min and the supernatant was collected; the mixture was then filtered tangentially to obtain the pomegranate exosomes.

[0111] Preparation Example 4

[0112] This preparation example provides a pomegranate exosome, which differs from Preparation Example 1 only in that: polygalacturonase is not added, and its reduced amount is proportionally allocated to arabinosidase and β-glucosidase, while the other raw materials and steps remain unchanged.

[0113] Preparation Example 5

[0114] This preparation example provides a pomegranate exosome, which differs from Preparation Example 1 only in that: arabinosidase is not added, and its reduced amount is proportionally allocated to polygalacturonase and β-glucosidase, while the other raw materials and steps remain unchanged.

[0115] Preparation Example 6

[0116] This preparation example provides a pomegranate exosome, which differs from Preparation Example 1 only in that: β-glucosidase is not added, and its reduced amount is proportionally allocated to polygalacturonase and arabinosidase, while the other raw materials and steps remain unchanged.

[0117] Preparation Example 7

[0118] This preparation example provides a pomegranate exosome, which is prepared by a method including the following steps:

[0119] Add 100 g of pomegranate fruit to 200 mL of PBS buffer and crush thoroughly to obtain a mixture. Centrifuge the mixture at 3000 g for 10 min and collect the supernatant; then centrifuge at 8000 g for 20 min and collect the supernatant; then centrifuge at 10000 g for 60 min and collect the supernatant; filter tangentially to obtain the pomegranate exosomes.

[0120] Preparation Example 8

[0121] This preparation example provides a pullulan polysaccharide, which is prepared by a method comprising the following steps:

[0122] 10 g of citrus, 0.3 g of protease, 0.2 g of laccase, and 0.4 g of fruit acid enzyme were added to water and mixed thoroughly. The mixture was reacted at 40℃ for 30 min, followed by enzyme inactivation at 120℃ for 80 min. The mixture was then concentrated and dried to obtain the citrus enzymatic hydrolysate. Activated *Brachystomata* buddingus was inoculated into a basal medium containing 10 wt% citrus enzymatic hydrolysate. The initial viable cell count of the fermentation system was 3 × 10⁻⁶. 9 The extract was fermented at 30℃ for 25 h at CFU / mL, then centrifuged at 11000 rpm for 15 min. The supernatant was collected, and ethanol with a volume twice that of the supernatant was added. The precipitate was collected, washed three times, and dried to obtain pullulan polysaccharide.

[0123] Preparation Example 9

[0124] This preparation example provides a pullulan polysaccharide, which is prepared by a method comprising the following steps:

[0125] 10 g of citrus, 0.2 g of protease, 0.4 g of laccase, and 0.4 g of fruit acid enzyme were added to water and mixed thoroughly. The mixture was reacted at 45°C for 20 min, followed by enzyme inactivation at 130°C for 5 min. The mixture was then concentrated and dried to obtain the citrus enzymatic hydrolysate. Activated *Brachystomata* buddingus was inoculated into a basal medium containing 15 wt% citrus enzymatic hydrolysate. The initial viable cell count of the fermentation system was 5 × 10⁻⁶. 9 The extract was fermented at 27℃ for 30 h at CFU / mL, then centrifuged at 10000 rpm for 20 min. The supernatant was collected, and ethanol (3 times the volume of the supernatant) was added. The precipitate was collected, washed 3 times, and dried to obtain pullulan polysaccharide.

[0126] Preparation Example 10

[0127] This preparation example provides a pullulan polysaccharide, which is prepared by a method comprising the following steps:

[0128] 10 g of citrus, 0.1 g of protease, 0.1 g of laccase, and 0.1 g of fruit acid enzyme were added to water and mixed thoroughly. The mixture was reacted at 35°C for 40 min, followed by enzyme inactivation at 110°C for 10 min. The mixture was then concentrated and dried to obtain the citrus enzymatic hydrolysate. Activated *Brachystomata* buddingus was inoculated into a basal medium containing 5 wt% citrus enzymatic hydrolysate. The initial viable cell count of the fermentation system was 1 × 10⁻⁶. 9 The extract was fermented at 35℃ for 20 h at CFU / mL, then centrifuged at 12000 rpm for 10 min. The supernatant was collected, and 1.5 times the volume of ethanol was added to collect the precipitate. The precipitate was washed three times and dried to obtain pullulan polysaccharide.

[0129] Preparation Example 11

[0130] This preparation example provides a pullulan polysaccharide, which differs from Preparation Example 8 only in that: no protease is added, and its reduced amount is proportionally allocated to laccase and fruit acid enzyme, while the other raw materials and steps remain unchanged.

[0131] Preparation Example 12

[0132] This preparation example provides a pullulan polysaccharide that differs from Preparation Example 8 only in that: laccase is not added, and its reduced amount is proportionally allocated to the protease and fruit acid enzyme, while the other raw materials and steps remain unchanged.

[0133] Preparation Example 13

[0134] This preparation example provides a pullulan polysaccharide, which differs from Preparation Example 8 only in that: no fruit acid enzyme is added, and its reduced amount is proportionally allocated to protease and laccase, while the other raw materials and steps remain unchanged.

[0135] Preparation Example 14

[0136] This preparation example provides a pullulan polysaccharide, which is prepared by a method comprising the following steps:

[0137] Activated budding short-circuit fungi were inoculated into the basal medium, and the initial viable count of the fermentation system was 3 × 10⁻⁶. 9 The extract was fermented at 30℃ for 25 h at CFU / mL, then centrifuged at 11000 rpm for 15 min. The supernatant was collected, and ethanol with a volume twice that of the supernatant was added. The precipitate was collected, washed three times, and dried to obtain pullulan polysaccharide.

[0138] Example 1

[0139] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0140] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8The pomegranate exosomes provided in Preparation Example 1 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 8, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0141] Example 2

[0142] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0143] An aqueous solution containing 1 mg of nicotine and 1×10 9 The pomegranate exosomes provided in Preparation Example 2 were mixed and sonicated at 300 W for 15 min to obtain an exosome complex. By weight, 10 parts pullulan from Preparation Example 9, 2 parts peppermint oil, 5 parts β-sitosterol, and 85 parts water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 15:1 to obtain the nicotine gel.

[0144] Example 3

[0145] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0146] An aqueous solution containing 0.1 mg nicotine and 1×10 8 The pomegranate exosomes provided in Preparation Example 3 were mixed and sonicated at 100 W for 30 min to obtain an exosome complex. A gel matrix was prepared by mixing 1 part pullulan from Preparation Example 10, 0.1 part menthol, 1 part hyaluronic acid, and 70 parts water by weight. The gel matrix and the exosome complex were mixed at a mass ratio of 10:3 to obtain the nicotine gel.

[0147] Example 4

[0148] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0149] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The pomegranate exosomes provided in Preparation Example 4 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 8, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0150] Example 5

[0151] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0152] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The pomegranate exosomes provided in Preparation Example 5 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 8, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0153] Example 6

[0154] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0155] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The pomegranate exosomes provided in Preparation Example 6 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 8, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0156] Example 7

[0157] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0158] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The pomegranate exosomes provided in Preparation Example 7 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 8, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0159] Example 8

[0160] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0161] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8The pomegranate exosomes provided in Preparation Example 1 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 11, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0162] Example 9

[0163] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0164] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The pomegranate exosomes provided in Preparation Example 1 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 12, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0165] Example 10

[0166] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0167] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The pomegranate exosomes provided in Preparation Example 1 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 13, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0168] Example 11

[0169] This embodiment provides a nicotine gel, which is prepared by a method including the following steps:

[0170] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The pomegranate exosomes provided in Preparation Example 1 were mixed and sonicated at 200 W for 20 min to obtain an exosome complex. Five parts by weight of pullulan from Preparation Example 14, one part of menthol, three parts of ceramide NP, and 80 parts of water were mixed to prepare a gel matrix. The gel matrix and the exosome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0171] Comparative Example 1

[0172] This comparative example provides a nicotine gel, which is prepared by a method comprising the following steps:

[0173] 150 mg of soybean lecithin, 25 mg of cholesterol, 10 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000 were mixed and dissolved with 100 mL of ethanol. The organic solvent was then removed by evaporation at 40 °C to form a thin film. PBS buffer was added and stirred to form liposomes.

[0174] An aqueous solution containing 0.5 mg nicotine and 5 × 10 8 The liposomes were mixed and sonicated at 200 W for 20 min to obtain a liposome complex. By weight, 5 parts pullulan from Preparation Example 8, 1 part menthol, 3 parts ceramide NP, and 80 parts water were mixed to prepare a gel matrix. The gel matrix and the liposome complex were mixed at a mass ratio of 13:2 to obtain the nicotine gel.

[0175] Test Example 1

[0176] SD mouse skin was fixed between the supply and receiving pools of a transdermal device, with the stratum corneum facing the supply pool. 0.9% physiological saline receiving solution was added, and air bubbles were removed to ensure complete contact between the receiving solution surface and the inner layer of the skin. After equilibration, the receiving solution was replaced with fresh solution. Two g of the nicotine gel provided in Examples 1-11 and Comparative Example 1 were evenly applied to the mouse skin. This time was defined as 0 h. Samples were taken at fixed time points thereafter, and an equal volume of fresh receiving solution was added periodically. All samples were processed and analyzed according to the chromatographic conditions of the methodology. Peak areas were measured, and a standard curve solution was prepared using the external standard method for quantitative analysis.

[0177] As shown in Table 1, Examples 1-3 and Comparative Example 1 demonstrate that the phospholipid bilayer of pomegranate exosomes can fuse with skin cells, enabling nicotine penetration while simultaneously delivering active ingredients such as proteins and RNA carried within to the deeper layers of the skin. By influencing the skin microenvironment, this indirectly improves skin permeability, thereby regulating the transdermal nicotine transdermal rate. Example 7 shows that enzyme-assisted extraction of pomegranate exosomes can stabilize exosome activity, thus affecting the transdermal nicotine transdermal rate. Examples 4-6 demonstrate that polygalacturonase, arabinosidase, and β-glucosidase have a synergistic effect, ensuring the integrity and activity of pomegranate exosomes, thereby influencing the transdermal nicotine transdermal rate. Example 11 shows that adding citrus enzymatic hydrolysate during the fermentation of *Brachystomata buddingii* can improve the molecular weight and glycosidic bond ratio of pullulan, affecting the three-dimensional network structure of the pullulan gel matrix and resulting in a better regulatory effect on the transdermal nicotine transdermal rate. As shown in Examples 8-10, protease, laccase, and fruit acid enzyme have a synergistic effect. The citrus enzymatic hydrolysate prepared by the combination affects the fermentation and metabolism of *Brachystomata buddingis*, thereby affecting the structure of pullulan polysaccharide gel matrix and having a better regulatory effect on nicotine transdermal absorption.

[0178]

[0179] Test Example 2

[0180] The fur on the back of the rabbits was clipped using an electric razor. Four healthy skin sites were selected as administration sites, two on each side of the back, centered on the midline of the back. Two g of the nicotine gel provided in Examples 1-11 and Comparative Example 1 were evenly applied to each administration site using a scraper. The sites were then secured with non-woven adhesive bandages, wrapped with gauze, and finally sealed with an elastic adhesive bandage. Three rabbits were used in each group, with four administration sites per rabbit, for a total of 12 administration sites. The coverings and nicotine gel were removed after 24 h, 48 h, and 72 h of administration. Skin reactions were observed visually after 30 min. Skin reactions were evaluated according to the criteria shown in Table 2. A skin reaction score (total score for erythema / scab formation and edema formation) was calculated for each administration site. The Primary Irritation Index (PII) was calculated, and irritation was evaluated according to the criteria shown in Table 3. Primary Stimulation Index (PII) = (average skin reaction scores at each administration site over 24 hours + average skin reaction scores at each administration site over 48 hours + average skin reaction scores at each administration site over 72 hours) / 3.

[0181] As shown in Table 4, Examples 1-3 and Comparative Example 1 demonstrate that the phospholipid bilayer of pomegranate exosomes can fuse with skin cells, enabling nicotine penetration while simultaneously delivering the active ingredients such as proteins and RNA carried within to the deeper layers of the skin, thus reducing the skin irritation caused by nicotine. Example 7 shows that enzyme-assisted extraction of pomegranate exosomes can stabilize exosome activity and reduce the skin irritation caused by nicotine. Examples 4-6 demonstrate that polygalacturonase, arabinosidase, and β-glucosidase have a synergistic effect, ensuring the integrity and activity of pomegranate exosomes, thereby reducing the skin irritation caused by nicotine. Example 11 shows that adding citrus enzymatic hydrolysate during the fermentation of *Brachystomata buddingii* can improve the molecular weight and glycosidic bond ratio of pullulan, reducing the skin irritation caused by nicotine. As shown in Examples 8-10, protease, laccase, and fruit acid enzyme have a synergistic effect. The citrus enzymatic hydrolysate prepared by the combination affects the fermentation and metabolism of *Brachystomata buddingii*, thereby affecting the structure of pullulan polysaccharide and reducing the skin irritation of nicotine.

[0182]

[0183]

[0184]

[0185] This invention illustrates a low-irritant nicotine gel, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials in the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0186] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0187] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A low-irritant nicotine gel, characterized in that, The nicotine gel includes a gel matrix and an exosome complex embedded in the gel matrix; The components of the gel matrix include pullulan and water; The exosome complex includes pomegranate exosomes and nicotine loaded in pomegranate exosomes.

2. The nicotine gel according to claim 1, characterized in that, The mass ratio of the gel matrix to the exosome complex is (10-15):(1-3); Preferably, the components of the gel matrix include 1-10 parts pullulan and 70-85 parts water by weight; Preferably, the ratio of pomegranate exosomes to nicotine is (1-10)×10⁻⁶. 8 Quantity: (0.01-1) mg.

3. The nicotine gel according to claim 1 or 2, characterized in that, The pomegranate exosomes were prepared by a method comprising the following steps: Pomegranate fruit was mixed with buffer solution, crushed, and then mixed with coenzyme for enzymatic hydrolysis. The supernatant was collected by differential centrifugation and purified to obtain the pomegranate exosomes.

4. The nicotine gel according to claim 3, characterized in that, The ratio of pomegranate fruit to buffer solution is 1 g:(1.5-3) mL; Preferably, the buffer solution comprises any one or a combination of at least two of PBS buffer, HEPES buffer, or Tris-HCl buffer; Preferably, the mass ratio of pomegranate fruit to coenzyme is 1:(0.001-0.01); Preferably, the coenzyme includes any one or a combination of at least two of polygalacturonase, arabinosidase, or β-glucosidase; Preferably, the coenzyme includes polygalacturonase, arabinosidase, and β-glucosidase; Preferably, the mass ratio of polygalacturonase, arabinosidase and β-glucosidase is (1-3):(1-5):(1-8); Preferably, the enzymatic hydrolysis reaction is carried out at a temperature of 30-40°C for 1-3 hours. Preferably, the differential centrifugation includes three centrifugations: the first centrifugation is performed at a speed of 2000-4000 g for 5-15 min; the second centrifugation is performed at a speed of 7000-9000 g for 15-25 min; and the third centrifugation is performed at a speed of 9000-11000 g for 55-65 min. Preferably, the purification method includes tangential flow filtration.

5. The nicotine gel according to any one of claims 1-4, characterized in that, The pullulan polysaccharide was prepared by a method comprising the following steps: Citrus, extractant, and water are mixed and subjected to enzymatic hydrolysis to inactivate the enzyme and obtain citrus hydrolysate. *Bacillus buddingus* is inoculated into a culture medium containing the citrus hydrolysate and fermented. After centrifugation, the supernatant is collected, mixed with ethanol, and the precipitate is collected to obtain pullulan polysaccharide.

6. The nicotine gel according to claim 5, characterized in that, The mass ratio of the citrus fruit to the extracting enzyme is 1:(0.01-0.1); Preferably, the extraction enzyme includes any one or a combination of at least two of protease, laccase, or fruit acid enzyme; Preferably, the extraction enzyme includes protease, laccase, and fruit acid enzyme; Preferably, the mass ratio of the protease, laccase, and fruit acid enzyme is (1-8):(1-5):(1-5); Preferably, the enzymatic hydrolysis reaction is carried out at a temperature of 35-45°C for a time of 20-40 min. Preferably, the enzyme inactivation temperature is 110-130℃ and the time is 5-10 min; Preferably, the enzyme inactivation process further includes a concentration and drying step; Preferably, the viable count of the budding short-stem mold at the start of the fermentation system is (1-5)×10⁻⁶. 9 CFU / mL; Preferably, the citrus enzymatic hydrolysate has a mass percentage of 5-15 wt% in the culture medium. Preferably, the culture medium includes a carbon source, a nitrogen source, inorganic salts, and water; Preferably, the fermentation temperature is 27-35℃ and the time is 20-30 h; Preferably, the centrifugation speed is 10000-12000 rpm and the time is 10-20 min; Preferably, the volume ratio of the supernatant to ethanol is 1:(1.5-3); Preferably, the collection of sediment further includes a washing and / or drying step.

7. The nicotine gel according to any one of claims 1-6, characterized in that, The components of the gel matrix also include a cooling agent; Preferably, the components of the gel matrix further include 0.1-2 parts by weight of a cooling agent; Preferably, the cooling agent comprises any one or a combination of at least two of menthol, peppermint oil, menthol, menthone, ethyl menthane formamide, menthyl acetate, or limonene; Preferably, the combination of the gel matrix further includes a humectant; Preferably, the components of the gel matrix further include 1-5 parts by weight of a humectant; Preferably, the moisturizer comprises any one or a combination of at least two of the following: ceramide, β-sitosterol, hyaluronic acid, trehalose, hydroxyethyl urea, asiaticoside, panthenol, or allantoin.

8. The method for preparing nicotine gel according to any one of claims 1-7, characterized in that, The method includes: Pomegranate exosomes and nicotine were mixed and sonicated to obtain the exosome complex; pullulan was mixed with water to prepare a gel matrix; the gel matrix and the exosome complex were mixed to obtain the nicotine gel.

9. The method according to claim 8, characterized in that, The preparation of the gel matrix also includes the addition of cooling agents and / or moisturizers; Preferably, the power of the ultrasound is 100-300 W and the duration is 15-30 min.

10. The application of the nicotine gel according to any one of claims 1-7 in reducing nicotine dependence, characterized in that, The nicotine gel is used by applying it to the skin.

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