Zanthoxylum bungeanum volatile oil nanoemulsion gel, and preparation method and application thereof

Abstract

This invention discloses a nanoemulsion gel containing Sichuan pepper volatile oil, its preparation method, and its application. The Sichuan pepper volatile oil nanoemulsion gel of this invention comprises a core layer and an encapsulation layer. The core layer is a nanoemulsion loaded with Sichuan pepper volatile oil, and the encapsulation layer is a carboxymethyl chitosan-sodium alginate composite hydrogel. The composite hydrogel forms a three-dimensional network structure through calcium ion cross-linking, encapsulating the Sichuan pepper volatile oil-loaded nanoemulsion within it. This invention prepares Sichuan pepper volatile oil into a nanoemulsion, significantly improving its water solubility and chemical stability, and reducing degradation in the gastrointestinal tract after oral administration. Further encapsulation with the carboxymethyl chitosan-sodium alginate composite hydrogel utilizes the pH sensitivity and colonic enzyme responsiveness of the composite hydrogel to reduce drug release in the stomach and small intestine, allowing the drug to degrade and release upon reaching the colon, thus increasing drug accumulation at sites of colonic inflammation. This solves the problems of poor targeting and low bioavailability of Sichuan pepper volatile oil.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to a Sichuan pepper volatile oil nanoemulsion gel, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease with an incompletely defined etiology. The lesions are primarily confined to the mucosa and submucosa of the rectum and colon, exhibiting a continuous and diffuse distribution. Clinically, it is characterized by recurrent diarrhea, paroxysmal abdominal pain, and bloody, mucoid stools, often accompanied by systemic manifestations such as malnutrition and anemia. It is listed by the World Health Organization as one of the clinically refractory diseases. The disease has a global distribution, with high incidence rates in Europe and North America, reaching 24.3 / 100,000 annually and 19.2 / 100,000 annually, respectively. In my country and other Asian countries, the incidence rate has shown a significant upward trend in recent years, peaking in young adults aged 20-49. The male-to-female ratio is close to 1.0:1-1.3, severely impacting patients' quality of life. Severe cases are prone to fatal complications such as toxic megacolon and intestinal perforation, with a mortality rate as high as 44% for toxic megacolon and an incidence of spontaneous intestinal perforation of approximately 2%, posing a significant challenge to clinical diagnosis and treatment.

[0003] Current clinical treatment of ulcerative colitis (UC) focuses on controlling inflammation, relieving symptoms, and preventing relapse. Commonly used drugs include aminosalicylic acid preparations, glucocorticoids, and immunosuppressants. Although these drugs can play a role in controlling symptoms during the acute phase, they generally suffer from poor therapeutic selectivity. After administration, these drugs are widely distributed throughout the body, making it difficult to achieve effective therapeutic concentrations at the site of colonic lesions, resulting in limited clinical efficacy. Furthermore, long-term use can easily lead to serious side effects—for example, immunosuppressants can induce gastrointestinal reactions and bone marrow suppression, and glucocorticoids are only suitable for inducing remission during the active phase; long-term use can easily lead to dependence and significant adverse reactions, failing to meet the long-term treatment needs of UC patients. Therefore, the development of novel therapeutic agents with strong targeting, high bioavailability, and good safety has become an urgent need in UC clinical research.

[0004] Zanthoxylum Bungeanum Essential Oil (ZBEO) is a volatile active ingredient extracted from the fruit of plants in the Rutaceae family. Its composition is complex and rich in activity. Besides limonene, linalool, and lauric acid, it also contains key anti-inflammatory components such as hydroxy-α-sanshool. Limonene has been certified by the US FDA to relieve gastrointestinal spasms, and hydroxy-α-sanshool has an inhibition rate of up to 78.3% on chronic inflammation. Modern pharmacological studies have confirmed that ZBEO exerts its pharmacological effects through multiple mechanisms: it not only significantly inhibits the release of inflammatory factors and reduces intestinal mucosal inflammation, but also inhibits common intestinal pathogens such as Staphylococcus aureus and Escherichia coli. Simultaneously, it possesses antioxidant activity and intestinal flora regulation functions, which can improve the disordered intestinal microecological environment of UC patients, and has potential application value in the treatment of UC. However, the inherent defects of ZBEO severely limit its clinical translation: First, it has extremely poor water solubility, making it difficult to form a uniform dispersion system in the gastrointestinal tract; second, it has weak chemical stability, the active ingredients are easily volatile, and the activity can decrease by 50% after being left at room temperature for three days, and it is easily degraded by gastrointestinal digestive enzymes after oral administration; third, it has a significant first-pass effect in the liver, which further reduces the bioavailability of the drug, making it difficult for it to exert an effective therapeutic effect on the lesions in the colon.

[0005] Oral administration has become the preferred route of administration for the treatment of ulcerative colitis (UC) due to its advantages such as ease of operation, non-invasiveness, flexible dosage adjustment, and high patient compliance. However, the lesions of UC are mainly located in the colon, and oral drugs must cross complex gastrointestinal physiological barriers to reach the target area, facing multiple challenges: there is a significant pH gradient in the gastrointestinal tract (pH 1-2 in the stomach, pH 7-8 in the colon), and the highly acidic gastric juice can easily destroy the drug structure; the gastrointestinal mucus layer is composed of a three-dimensional network structure of mucin with pore sizes between 100 and 500 nm, which can trap drug particles through size sieving effect, while the dynamic renewal characteristics of the mucus layer accelerate drug clearance; in addition, the abundant digestive enzyme system in the gastrointestinal tract can degrade the active ingredients of the drug, and the non-specific distribution of the drug in the upper gastrointestinal tract ultimately results in the local drug concentration in the colonic lesion area being far below the effective therapeutic level, seriously affecting the drug administration effect.

[0006] To overcome the bottlenecks in oral drug delivery, nanodelivery systems and polysaccharide hydrogel carriers have become research hotspots. Nanoemulsions, as novel lipid carriers, can construct uniformly dispersed colloidal systems through the rational ratio of oil, aqueous, and emulsifier phases, effectively improving the solubility and chemical stability of lipid-soluble drugs, reducing drug degradation by gastrointestinal enzymes, and allowing their particle size to be controlled below 200 nm, facilitating penetration through the pores of the mucus layer. However, nanoemulsions alone lack colonic targeting; after oral administration, their structure is easily disrupted in the acidic and enzymatic environment of the stomach and small intestine, leading to premature drug leakage and preventing targeted release into the colon.

[0007] Polysaccharide hydrogels offer a novel approach to addressing these challenges due to their excellent biocompatibility, pH sensitivity, and colon-targeting properties. They maintain structural stability in the stomach and small intestine, protecting the encapsulated drug from release, and upon reaching the colon, they can be degraded by glycosidases produced by intestinal flora, achieving targeted drug delivery. Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan, possesses excellent biocompatibility, degradability, and metal ion adsorption capacity. Its abundant amino and carboxyl groups can coordinate with inorganic ions to form hydrogels. Sodium alginate (SA), a natural anionic polysaccharide, can rapidly form a stable hydrogel under calcium ion cross-linking. This hydrogel shrinks in the acidic environment of the stomach, further reducing drug leakage, while it is easily degraded in the neutral environment of the colon, promoting controlled drug release. Studies have shown that the composite hydrogel formed by the polyelectrolyte interaction of carboxymethyl chitosan and sodium alginate can optimize colon-targeting performance through the synergistic effect of both, effectively improving drug retention and release efficiency in the colon.

[0008] Based on the aforementioned research background and technical bottlenecks, this invention innovatively constructs a dual delivery system of "nanoemulsion-composite hydrogel": firstly, ZBEO is loaded using nanoemulsion technology to address its poor water solubility and weak stability; then, the ZBEO nanoemulsion is further encapsulated using the hydrophilic three-dimensional structure of a carboxymethyl chitosan-sodium alginate composite hydrogel. Utilizing the pH sensitivity and colonic enzyme responsiveness of the composite hydrogel, the nanoemulsion is protected from destruction in the stomach and small intestine, achieving precise drug delivery to the lesion site in the colon. This formulation simultaneously solves the core problems of low oral bioavailability and poor targeting of ZBEO, providing a safe, efficient, and well-compliant novel oral formulation for the clinical treatment of ulcerative colitis. Summary of the Invention

[0009] To address the issues of significant side effects and imprecise drug release in existing UC treatments, as well as the poor water solubility and low bioavailability of Sichuan pepper volatile oil when applied directly, this invention prepares a Sichuan pepper volatile oil nanoemulsion gel. This gel achieves targeted intestinal release of the drug, improves bioavailability, solves the problem of poor compliance, enhances the efficacy of UC relief, and reduces toxic side effects.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The Sichuan pepper volatile oil nanoemulsion gel of the present invention comprises a core layer and an encapsulation layer. The core layer is a nanoemulsion loaded with Sichuan pepper volatile oil, and the encapsulation layer is a carboxymethyl chitosan-sodium alginate composite hydrogel. The composite hydrogel forms a three-dimensional network structure through calcium ion cross-linking, thereby encapsulating the Sichuan pepper volatile oil-loaded nanoemulsion therein.

[0011] The specific steps of the preparation method of the Sichuan pepper volatile oil nanoemulsion gel of the present invention are as follows: (1) Preparation of nanoemulsion loaded with Sichuan pepper volatile oil: The volatile oil of Sichuan pepper, emulsifier, and co-emulsifier were mixed evenly to obtain an oil phase mixture. Under stirring conditions, ultrapure water was slowly added to the oil phase until the solution system became clear, transparent, and slightly bluish-white, yielding a nanoemulsion loaded with Sichuan pepper volatile oil. The mass concentration of Sichuan pepper volatile oil in the nanoemulsion was 2-10%, the mass concentration of the emulsifier was 7.5-13.5%, and the mass concentration of the co-emulsifier was 2.5-4.5%. (2) Preparation of carboxymethyl chitosan-sodium alginate composite hydrogel-encapsulated nanoemulsions: Carboxymethyl chitosan aqueous solution and sodium alginate aqueous solution were prepared separately with a mass concentration of 1-3%. The two solutions were mixed evenly at a mass ratio of 1:0.5-2 to obtain a composite hydrogel matrix solution. The nanoemulsion loaded with Sichuan pepper volatile oil prepared in step (1) was added to the composite hydrogel matrix solution at a mass ratio of 1:2-12. The mixture was stirred to disperse it evenly. After stirring was stopped and the bubbles were eliminated, calcium chloride aqueous solution was added dropwise to the above mixture under stirring. The addition of calcium chloride was stopped when the mass ratio of calcium chloride to the mixture was 1:18-19. The mixture was allowed to stand until it formed a gel to obtain Sichuan pepper volatile oil nanoemulsion gel.

[0012] In step (1), the emulsifier is one or more of Tween 80, Tween 85, soybean lecithin, and polyoxyethylene castor oil, preferably polyoxyethylene castor oil, and the co-emulsifier is one or more of glycerin, PEG400, anhydrous ethanol, and glycerol, preferably anhydrous ethanol.

[0013] In step (1), the stirring speed is 1000rpm-1200rpm and the stirring time is 5-10min, preferably 1000rpm and 5min.

[0014] In step (1), the preferred mass concentration of Sichuan pepper volatile oil in the nanoemulsion is 8%, the mass concentration of emulsifier is 9%, and the mass concentration of co-emulsifier is 3%.

[0015] In step (2), the preferred mass concentration of the carboxymethyl chitosan aqueous solution is 2%, the mass concentration of the sodium alginate aqueous solution is 2%, and the mass ratio of the two solutions is 1:1.

[0016] In step (2), the preferred mass ratio of the nanoemulsion loaded with Sichuan pepper volatile oil to the composite hydrogel matrix solution is 1:2. The nanoemulsion is added to the composite hydrogel matrix solution and magnetically stirred for 10-30 minutes to disperse it evenly.

[0017] In step (2), the mass concentration of the calcium chloride aqueous solution is 3%, the crosslinking temperature is room temperature, and the stirring speed is 1000-1500 rpm.

[0018] In step (2), the gelation time is determined by tilting the sample bottle. Timing starts from the first drop of calcium chloride solution is added. After the addition of calcium chloride is stopped, the sample bottle is tilted and the flow of the solution is observed. Timing is stopped when the system stops flowing when the sample bottle is tilted. This time is the gelation time.

[0019] The Sichuan pepper volatile oil nanoemulsion gel of the present invention can be used to prepare a drug for relieving ulcerative colitis.

[0020] The Sichuan pepper volatile oil nanoemulsion gel obtained by this invention is soft and smooth, injectable, does not separate into layers, and has uniform and stable properties.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention prepares Sichuan pepper volatile oil into a nanoemulsion, significantly improving its water solubility and chemical stability, and reducing degradation in the gastrointestinal tract after oral administration. It is then encapsulated in a carboxymethyl chitosan-sodium alginate composite hydrogel. Utilizing the pH sensitivity and colonic enzyme responsiveness of the composite hydrogel, the release of the drug in the stomach and small intestine is reduced. Upon reaching the colon, the drug degrades and releases, increasing its concentration at sites of colonic inflammation. This solves the problems of poor targeting and low bioavailability of Sichuan pepper volatile oil.

[0022] 2. Carboxymethyl chitosan is a water-soluble derivative of chitosan, containing amino and carboxyl active groups. Sodium alginate is a natural polysaccharide. Both have the advantages of good biocompatibility, in vivo degradation and absorption characteristics, and no toxic side effects. The three-dimensional network structure formed by sodium alginate under the action of calcium ions can stably encapsulate nanoemulsions. The synergistic effect of the two enhances the targeted therapeutic effect of the formulation.

[0023] 3. The anti-inflammatory and antibacterial effects of Sichuan pepper volatile oil are combined with the function of the composite hydrogel in regulating intestinal flora and protecting the intestinal mucosal barrier to achieve synergistic effects. It can significantly alleviate symptoms such as weight loss and colon shortening in UC mice, reduce colon tissue damage, reduce the level of inflammatory factors, regulate the composition of intestinal flora, and increase the abundance of beneficial bacteria. Moreover, the efficacy is better than using Sichuan pepper volatile oil or nanoemulsion alone, and the formulation is safe.

[0024] 4. The preparation process of this invention is simple and mild, the operation is controllable, no complicated equipment is required, the production cost is low, and it does not change the main components of Sichuan pepper volatile oil that exert its medicinal effect. Attached Figure Description

[0025] Figure 1 Schematic diagram of the preparation of oral Sichuan pepper volatile oil nanoemulsion gel and its effect on relieving colitis.

[0026] Figure 2 Total ion chromatogram of the volatile oil of Sichuan pepper prepared in Example 1.

[0027] Figure 3Transmission electron microscopy (TEM) image (A) of the nanoemulsion loaded with Sichuan pepper volatile oil prepared in Example 2 and scanning electron microscopy image (B) of the Sichuan pepper volatile oil nanoemulsion gel.

[0028] Figure 4 Stability study of the pepper volatile oil nanoemulsion prepared in Example 2: (A) Changes in nanoemulsion particle size before and after centrifugation, (B) Changes in nanoemulsion particle size and PDI at 37℃.

[0029] Figure 5 : Appearance of hydrogel matrices of different concentrations in Comparative Example 3; SxCyCaz represents the blank matrix formed by x×10 mg / ml sodium alginate, y×10 mg / ml carboxymethyl chitosan, and z×10 mg / ml calcium chloride solution.

[0030] Figure 6 Mechanical properties of the hydrogel matrix in different pH solutions in Comparative Example 3; (A) Hardness of the gel matrix in a buffer solution at pH 1.5; (B) Hardness of the gel matrix in a buffer solution at pH 6.5; (C) Hardness of the gel matrix in a buffer solution at pH 7.4; (D) Viscosity of the gel matrix in a buffer solution at pH 1.5; (E) Viscosity of the gel matrix in a buffer solution at pH 6.5; (F) Viscosity of the gel matrix in a buffer solution at pH 7.4.

[0031] Figure 7 Schematic diagram of the modeling and treatment process of ulcerative colitis in mice.

[0032] Figure 8 Biocompatibility evaluation diagram of Sichuan pepper volatile oil nanoemulsion gel (HE staining of major organs).

[0033] Figure 9 Evaluation of the efficacy of ZBEO-NEH in UC: (A) weight change, (B) DAI score, (C) spleen coefficient, (D) colon length histogram.

[0034] Figure 10 HE staining images of colon tissue sections from mice in different groups after treatment with different drugs.

[0035] Figure 11 Comparison of serum inflammatory factor levels in mice after different drug treatments (A: IL-1β, B: IL-6). Detailed Implementation Example 1

[0036] Extraction of ZBEO from Sichuan pepper This invention employs method A for the determination of volatile oils under General Chapter 2204 of the 2025 edition of the Chinese Pharmacopoeia. The method involves steam distillation. First, dried Sichuan pepper pericarps are ground into powder and soaked in 5 times their volume of water for 0.5 hours. Then, 1 L of ultrapure water is added to every 100 g of powder in a round-bottom flask, and the mixture is kept at a gentle boil for approximately 3 hours. Oil and water are separated using an oil-water separator. After standing for a short time, the volatile oil of Sichuan pepper is obtained. An appropriate amount of anhydrous sodium sulfate is added, and the mixture is dried and filtered through a 0.22 μm filter membrane. The oil is then stored at 4 °C in the dark for later use. The calculated ZBEO yield (%) is 2.54% (ml / g). Example 2

[0037] Preparation of nanoemulsion loaded with Sichuan pepper volatile oil (ZBEO-NE): Accurately weigh 0.50 g of each co-emulsifier (glycerol, PEG400, anhydrous ethanol, and glycerol) and place them separately in 2 mL centrifuge tubes. Add excess Sichuan pepper volatile oil to each centrifuge tube, vortex for 5 min, and sonicate for 4 h to aid dissolution. Let stand overnight. Then centrifuge at 8000 r / min for 20 min, take the supernatant, dilute with n-hexane to a suitable concentration, and measure the absorbance at 235 nm using a UV-Vis spectrophotometer to calculate the equilibrium solubility of Sichuan pepper volatile oil in each excipient. The results show that the equilibrium solubility of Sichuan pepper volatile oil is highest in anhydrous ethanol, therefore anhydrous ethanol is determined to be the optimal co-emulsifier.

[0038] Tween 80, Tween 85, soybean lecithin, and castor oil polyoxyethylene ether-40 (EL-40) were selected as candidate emulsifiers, with emulsification effect and system stability as the screening criteria. At room temperature, the above three candidate emulsifiers were thoroughly mixed with Sichuan pepper volatile oil at mass ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, respectively, and stirred until the solution was homogeneous. Then, ultrapure water was added dropwise using a pipette while continuously stirring until the solution returned to a clear, transparent, and non-layered homogeneous state. The results showed that castor oil polyoxyethylene ether-40 (EL-40) formed the largest nanoemulsion region, the solution was clear, transparent, and slightly bluish, and the system stability was the best. Therefore, EL-40 was determined to be the optimal emulsifier.

[0039] The Km value represents the mass ratio of emulsifier to co-emulsifier. The optimal emulsifier, EL-40, and the optimal co-emulsifier, anhydrous ethanol, were selected and mixed uniformly at mass ratios of 2:1, 3:1, and 4:1 to prepare a mixed emulsifier (Smix). Smix with different Km values ​​was mixed with Sichuan pepper volatile oil at mass ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9. After thorough stirring, ultrapure water was added dropwise while stirring continuously, and the formation and appearance of the nanoemulsion were observed. Using a pseudo-ternary phase diagram, the clarity, transparency, and viscosity of the nanoemulsion under different Km values ​​were investigated. The results showed that when the Km value was 3:1, the formed nanoemulsion was clear and transparent, had moderate viscosity, and exhibited the best stability; this was determined to be the optimal Km value.

[0040] Smix was prepared by uniformly mixing emulsifier EL-40 and co-emulsifier anhydrous ethanol at an optimal Km ratio of 3:1. Smix was then mixed with Sichuan pepper volatile oil at mass ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, resulting in a total solution mass of 1g. The mixture was placed on a magnetic stirrer, and ultrapure water was slowly added dropwise while continuously stirring until a homogeneous and transparent nanoemulsion system was formed. The prepared nanoemulsions were sealed in a light-proof container and left at room temperature for 8 days, during which the emulsion morphology was observed. Considering the particle size and polydispersity index (PDI) of the nanoemulsions, the morphology of the emulsions formed with different proportions of oil phase was observed after 8 days. The nanoemulsion with the most stable emulsion, no stratification, and high drug loading was selected. Finally, the nanoemulsion formulation was determined to be a combination of EL-40 and anhydrous ethanol with a Sichuan pepper volatile oil mass ratio of 6:4 for subsequent experiments.

[0041] The particle size of the pepper volatile oil nanoemulsion prepared in Example 2 was determined to be 40.31 ± 0.32 nm using a Malvern particle size analyzer. The morphology of the pepper volatile oil nanoemulsion was observed using a transmission electron microscope. Figure 3 As shown in (A), the nanoemulsion prepared by this invention is spherical. The encapsulation efficiency of the ZBEO nanoemulsion obtained by this invention is 96.63% ± 0.43%, and the drug loading rate is 7.04% ± 0.24%. Figure 4 As shown in (A) and (B), the stability of the prepared Sichuan pepper volatile oil nanoemulsion was investigated. After centrifugation, no stratification was observed in the nanoemulsion, and the particle size remained almost unchanged before and after centrifugation. The PDI was still less than 0.15 after centrifugation, indicating that the prepared nanoemulsion has centrifugal stability. After storage at 37℃ for 8 days, the particle size and PDI of the nanoemulsion did not change significantly, indicating that the prepared nanoemulsion has good storage stability. Example 3

[0042] Preparation of Sichuan pepper volatile oil nanoemulsion gel (ZBEO-NEH): Carboxymethyl chitosan, sodium alginate, and calcium chloride were prepared with mass fractions of 10 mg / ml, 20 mg / ml, and 30 mg / ml, respectively. Approximately 3.0 g of sodium alginate was weighed and placed in a sample bottle, followed by approximately 3.0 g of carboxymethyl chitosan. The sample bottle was placed on a magnetic stirrer at 1000 rpm and stirred for 10 minutes to ensure thorough mixing. Stirring was stopped, and the mixture was allowed to stand for a period of time to eliminate air bubbles. When the system was clear, transparent, and bubble-free, the nanoemulsion loaded with Sichuan pepper volatile oil prepared in step (1) was added to the composite hydrogel matrix solution at a mass ratio of 1:2-12. The mixture was magnetically stirred for 10-30 minutes to ensure uniform dispersion. Calcium chloride was then added dropwise while stirring until the flow rate of the solution in the sample bottle slowed down. The addition of calcium chloride was then stopped, and the mixture was allowed to stand until it gelled. The gelation time was determined using the tilting method. Timing started from the addition of the first drop of calcium chloride solution. After stopping the addition of calcium chloride, the sample bottle was tilted, and the flow of the solution was observed. Timing was stopped when the system stopped flowing when the sample bottle was tilted. This time is the gelation time.

[0043] Comparative Example 1: Nanoemulsion loaded with Sichuan pepper volatile oil (ZBEONE) (without composite hydrogel encapsulation) Nanoemulsions loaded with Sichuan pepper volatile oil were prepared according to the method in Example 2, without subsequent composite hydrogel encapsulation steps, and were directly used as samples for later use.

[0044] Comparative Example 2: Blank Nanoemulsion Gel (NEH) (without Sichuan pepper volatile oil) Except for the absence of Sichuan pepper volatile oil, the other operations were completely consistent with those in Examples 2 and 3, and blank nanoemulsion gels were prepared.

[0045] Comparative Example 3: Carboxymethyl chitosan-sodium alginate composite hydrogel (without nanoemulsion encapsulation) Without performing the nanoemulsion preparation as in Example 2, a blank composite hydrogel was prepared by directly mixing a 2% carboxymethyl chitosan aqueous solution and a 2% sodium alginate aqueous solution at a mass ratio of 1:1 and then adding the mixture dropwise into a 3% calcium chloride aqueous solution for crosslinking. Figure 5 As shown in the figure, SxCyCaz represents the blank matrix formed by x×10mg / ml sodium alginate, y×10mg / ml carboxymethyl chitosan, and z×10mg / ml calcium chloride solution. The preferred hydrogel matrix is ​​a homogeneous and stable semi-solid, free of bubbles and clumps, with a soft and smooth texture, injectability, moderate viscosity, and no stratification.

[0046] like Figure 5 As shown, S1C1Ca1 and S3C3Ca2 were not fully formed and still contained excess liquid. S3C1Ca3 exhibited obvious gel-like properties and numerous air bubbles. The remaining hydrogel matrices showed good appearance. The mechanical properties of the hydrogels were analyzed using a texture analyzer. The hydrogels were immersed in different pH values ​​to analyze their hardness and viscosity. Figure 6As shown in (A), when the gel matrix was immersed in a buffer solution at pH 1.5, the stiffness of all groups increased, which is beneficial for the gel to pass through the strong acidic environment of the stomach without being destroyed. Figure 6 As shown in (B) and (C), when the gel matrix was immersed in buffer solutions with pH 6.5 and pH 7.4, the matrix stiffness increased in two groups, while the stiffness decreased in the remaining groups. Figure 6 (D) When the gel matrix was immersed in a buffer solution with a pH of 1.5, the viscosity of all 6 groups of gel matrices decreased significantly, which facilitated the smooth passage of the gel through the highly acidic environment of the stomach without adhesion. Figure 6 As shown in (E), when the gel matrix was immersed in a buffer solution at pH 6.5, only the S2C2Ca3 group showed a significant increase in viscosity, indicating that this concentration of matrix is ​​conducive to adhesion to the inflammatory microacidic environment of ulcerative colitis. Figure 6 As shown in (F), when the gel matrix was immersed in a buffer solution at pH 7.4, the viscosity of four matrices increased. In summary, to ensure that the hydrogel can pass through the acidic environment of the stomach and degrade at sites of colonic inflammation, the S2C2Ca3 group was preferred. Example 4

[0047] Safety evaluation of Sichuan pepper volatile oil nanoemulsion gel formulation After drug administration, mice were sacrificed, and major organs (heart, liver, spleen, lung, and kidney) were collected for hematoxylin-eosin staining analysis. Results are as follows: Figure 8 As shown, the experimental group mice showed no obvious pathological damage or inflammatory lesions in their major organs and colon tissues, proving that the pepper volatile oil nanoemulsion gel of the present invention has good biocompatibility and no acute toxic side effects. Example 5

[0048] In vivo efficacy evaluation of Sichuan pepper volatile oil nanoemulsion gel 1. Establishment of a mouse model of ulcerative colitis: Seventy C57BL / 6 mice were acclimatized for 7 days and then randomly divided into 7 groups, including a normal control group (CON), a model group (MOD), a Sichuan pepper volatile oil group (ZBEO), a Sichuan pepper volatile oil nano-emulsion group (ZBEO-NE), a pure material group (NEH), a Sichuan pepper volatile oil nanoemulsion gel group (ZBEO-NEH), and a positive control group (5-ASA), with 10 mice in each group. Except for the normal control group, the mice in the other groups were allowed to drink 2.5% sodium dextran sulfate (DSS) aqueous solution for 7 days to establish an ulcerative colitis model.

[0049] During the experiment, mice in the CON group were given a normal diet and free access to water daily. From day 8 onwards, they were administered physiological saline by gavage daily for 7 days. Mice in the MOD group drank drinking water containing 2.5% DSS daily. From day 8 onwards, they were administered physiological saline by gavage daily for 7 days. Mice in the drug-treated groups drank drinking water containing 2.5% DSS daily. From day 8 onwards, they were administered the corresponding drug by gavage daily for 7 days. Mouse weight, water intake, and food intake were recorded daily.

[0050] 2. Drug treatment: Starting from day 8 after the modeling process, mice in each group were administered the drug once daily by gavage for 7 consecutive days. The normal control group and the model control group were given an equal volume of physiological saline.

[0051] 3. Efficacy Evaluation: After drug administration, mice were sacrificed, and changes in body weight and colon length were recorded and photographed. The Disease Activity Index (DAI) was calculated. Colon tissue was collected for HE staining to observe pathological damage. Serum levels of IL-6 and IL-1β were measured.

[0052] Weight results as follows Figure 9 As shown in (A), the body weight of mice in the CON group remained stable with a slight upward trend throughout the 14-day experimental period, maintaining a weight range of 23–26 g without significant fluctuations. Mice in the MOD group and each of the drug-treated groups (ZBEO, ZBEONE, NEH, ZBEONEH, and 5-ASA groups) showed a slight increase in body weight during the first 5 days of the modeling period (1–7 days), followed by a gradual decrease, reaching its lowest point on day 8. During the recovery period (7–14 days), the body weight of mice in the MOD group continued to decrease, followed by a slight rebound, maintaining a weight of approximately 20 g on day 14, significantly lower than other groups. The body weight of mice in each drug-treated group showed varying degrees of recovery after reaching its lowest point, with the ZBEONEH group showing the largest recovery, reaching approximately 25 g on day 14, not significantly different from the CON group (approximately 25 g), indicating that this group effectively alleviated the weight loss caused by the modeling process. DAI scores are as follows: Figure 9 As shown in (B), the DAI score of mice in the CON group remained close to 0 throughout the process, with no colitis-related pathological symptoms. The DAI scores of mice in the MOD group and each intervention group gradually increased as the modeling process progressed, reaching a peak around day 7. Subsequently, the DAI scores of each group gradually decreased, but the rate of decrease differed significantly between groups. The ZBEONEH group showed the fastest decrease in DAI score, dropping below 0.5 by day 14, the lowest among all modeling groups. Spleen coefficient ( Figure 9 C) and colon length ( Figure 9 D) The results also showed that the ZBEO-NEH group had a better treatment effect. HE staining results ( Figure 10The results showed that the MOD group exhibited disordered glandular structure, atrophy or disappearance of some crypts, and even crypt abscesses. The mucosa and submucosa showed extensive infiltration of inflammatory cells (lymphocytes, neutrophils, etc.), indicating a significant inflammatory response, including crypt necrosis, decreased goblet cells, edema, increased leukocyte infiltration, and irregular mucosal structure. These pathological features indicated successful UC model construction. The ZBEO-NEH group showed the most significant repair effect, with near-normal restoration of colonic mucosal epithelial integrity, clear crypt structure, and significantly reduced inflammatory cell infiltration in the mucosa and submucosa, with only mild tissue edema. ELISA results showed ( Figure 11 ZBEO-NEH administration can reduce the levels of IL-1β and IL-6 in UC mice.

[0053] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A nanoemulsion gel of Sichuan pepper volatile oil, characterized in that: It includes a core layer and an encapsulation layer. The core layer is a nanoemulsion loaded with Sichuan pepper volatile oil, and the encapsulation layer is a carboxymethyl chitosan-sodium alginate composite hydrogel. The composite hydrogel forms a three-dimensional network structure through calcium ion cross-linking, encapsulating the nanoemulsion loaded with Sichuan pepper volatile oil within it.

2. A method for preparing the Sichuan pepper volatile oil nanoemulsion gel as described in claim 1, characterized in that: The specific steps of the method are as follows: (1) Preparation of nanoemulsion loaded with Sichuan pepper volatile oil: The volatile oil of Sichuan pepper, emulsifier, and co-emulsifier were mixed evenly to obtain an oil phase mixture. Under stirring conditions, ultrapure water was slowly added to the oil phase until the solution system became clear, transparent, and slightly bluish-white, yielding a nanoemulsion loaded with Sichuan pepper volatile oil. The mass concentration of Sichuan pepper volatile oil in the nanoemulsion was 2-10%, the mass concentration of the emulsifier was 7.5-13.5%, and the mass concentration of the co-emulsifier was 2.5-4.5%. (2) Preparation of carboxymethyl chitosan-sodium alginate composite hydrogel-encapsulated nanoemulsions: Carboxymethyl chitosan aqueous solution and sodium alginate aqueous solution were prepared separately with a mass concentration of 1-3%. The two solutions were mixed evenly at a mass ratio of 1:0.5-2 to obtain a composite hydrogel matrix solution. The nanoemulsion loaded with Sichuan pepper volatile oil prepared in step (1) was added to the composite hydrogel matrix solution at a mass ratio of 1:2-12. The mixture was stirred to disperse it evenly. After stirring was stopped and the bubbles were eliminated, calcium chloride aqueous solution was added dropwise to the above mixture under stirring. The addition of calcium chloride was stopped when the mass ratio of calcium chloride to the mixture was 1:18-19. The mixture was allowed to stand until it formed a gel to obtain Sichuan pepper volatile oil nanoemulsion gel.

3. The preparation method according to claim 2, characterized in that: In step (1), the emulsifier is one or more of Tween 80, Tween 85, soybean lecithin, and polyoxyethylene castor oil, and the co-emulsifier is one or more of glycerin, PEG400, anhydrous ethanol, and glycerol.

4. The preparation method according to claim 2, characterized in that: In step (1), the stirring speed is 1000rpm-1200rpm and the stirring time is 5-10min.

5. The preparation method according to claim 2, characterized in that: In step (1), the mass concentration of Sichuan pepper volatile oil in the nanoemulsion is 8%, the mass concentration of emulsifier is 9%, and the mass concentration of co-emulsifier is 3%.

6. The preparation method according to claim 2, characterized in that: In step (2), the mass concentration of the carboxymethyl chitosan aqueous solution is 2%, the mass concentration of the sodium alginate aqueous solution is 2%, and the mass ratio of the two solutions is 1:

1.

7. The preparation method according to claim 2, characterized in that: In step (2), the mass ratio of the nanoemulsion loaded with Sichuan pepper volatile oil to the composite hydrogel matrix solution is 1:

2. It is added to the composite hydrogel matrix solution and magnetically stirred for 10-30 minutes to disperse it evenly.

8. The preparation method according to claim 2, characterized in that: In step (2), the mass concentration of the calcium chloride aqueous solution is 3%, the crosslinking temperature is room temperature, and the stirring speed is 1000-1500 rpm.

9. The preparation method according to claim 2, characterized in that: In step (2), the gelation time is determined by tilting the sample bottle. Timing starts from the first drop of calcium chloride solution is added. After the addition of calcium chloride is stopped, the sample bottle is tilted and the flow of the solution is observed. Timing is stopped when the system stops flowing when the sample bottle is tilted. This time is the gelation time.

10. The use of the Sichuan pepper volatile oil nanoemulsion gel as described in claim 1 in the preparation of a medicament for relieving ulcerative colitis.

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