Anti-inflammatory analgesic microneedle patch loaded with natural pepper source double active components as well as preparation method and application of anti-inflammatory analgesic microneedle patch

Abstract

The invention relates to an anti-inflammatory analgesic microneedle patch loaded with natural pepper source double active components as well as a preparation method and application of the anti-inflammatory analgesic microneedle patch. The invention discloses a natural Chinese prickly ash essential oil nano-vesicle, which is prepared by compounding a natural Chinese prickly ash source nano-vesicle, a natural Chinese prickly ash essential oil nano-emulsion and a carrier matrix, the natural Chinese prickly ash source nano-vesicle is an exosome nano-vesicle extracted from Chinese prickly ash, the particle size of the natural Chinese prickly ash source nano-vesicle is 10-1000 nm, and the particle size of the natural Chinese prickly ash essential oil nano-emulsion is 50-500 nm; the natural zanthoxylum bungeanum essential oil nanoemulsion is prepared from zanthoxylum bungeanum waste obtained after natural zanthoxylum bungeanum source nano vesicles are extracted as a raw material. According to the invention, the two active components, namely the natural Chinese prickly ash source nano vesicles and the natural Chinese prickly ash essential oil nano emulsion, derived from the same plant are jointly integrated into the microneedle for the first time. The two nano-carriers not only improve the bioavailability of various bioactive substances in the Chinese prickly ash, but also regulate various inflammation signal channels at the same time through a complementary pharmacological mechanism, play a synergistic effect and are obviously superior to a single-component system.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials, transdermal drug delivery systems and natural medicine development technology, and in particular to an anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper, its preparation method and application. Background Technology

[0002] Transdermal drug delivery systems (TDDS), as an alternative to traditional oral or injectable drug delivery methods, have attracted much attention in the modern medical field due to their advantages such as avoiding drug degradation in the gastrointestinal tract and the first-pass effect in the liver, reducing systemic side effects, providing continuous and stable drug release, and improving patient compliance. However, the outermost layer of the skin, the stratum corneum, is a strong physical barrier that greatly limits the effective penetration of most drug molecules, thus affecting therapeutic efficacy.

[0003] The emergence of microneedles (MNs) technology offers an innovative solution to this challenge. Microneedles are arrays of hundreds of micrometer-sized needle tips that can penetrate the stratum corneum in a minimally invasive and painless manner, forming reversible microchannels in the skin. This allows for the efficient and precise delivery of drugs to the epidermis or dermis, significantly improving drug bioavailability. Among various microneedle materials, hydrogel microneedles based on natural polymers have become a research hotspot in tissue engineering and drug delivery due to their excellent biocompatibility, biodegradability, tunable mechanical properties, and ability to be rapidly molded through photopolymerization.

[0004] Chinese patent CN119454572A discloses a hydrogel microneedle patch, its preparation method, and its application. The patch uses a gel matrix obtained by mixing PEG600 and PEG1000 in a 3:4 mass ratio, loaded with erythromycin, *Vaccaria segetalis* flavonoid glycosides, and 7-demethylcortexin to prepare temperature-responsive hydrogel microneedles. At room temperature, the microneedles are solid needles that can penetrate bacterial biofilms under external force, then rapidly melt into a liquid at body temperature, releasing the loaded drugs. This method promotes wound healing through the synergistic effect between the hydrogel and the drugs. The prepared hydrogel microneedle patch significantly promotes rapid healing of Staphylococcus aureus-infected wounds and significantly promotes rapid healing of dorsal wounds in diabetic rats. This method uses conventional hydrogel microneedles based on natural polymers, loaded with erythromycin, *Vaccaria segetalis* flavonoid glycosides, and 7-demethylcortexin, each component contributing to different therapeutic effects.

[0005] Chinese patent CN118319837A discloses a microneedle slimming patch loaded with hydroxy-α-sanshool. It consists of hydroxy-α-sanshool as the active ingredient, a backing, and needle tips. The backing matrix is ​​prepared from one or more of polyvinyl alcohol (PVA), sodium carboxymethyl cellulose (CMC-Na), hydroxypropyl methylcellulose (HPMC), and methyl vinyl ether / maleic anhydride copolymer (PVM / MA). The needle tip matrix is ​​prepared from one or more of polyvinyl alcohol, polyvinylpyrrolidone (PVP), hyaluronic acid (HA), hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, and methyl vinyl ether / maleic anhydride copolymer. This soluble microneedle patch can easily carry hydroxy-α-sanshool through the stratum corneum of the skin, efficiently delivering the drug to the dermal capillaries for absorption, and then exerting its effects throughout the body via systemic circulation. The microneedle puncture process can stimulate a local inflammatory response in the skin, promoting metabolism and fat breakdown. Microneedle puncture causes less pain to patients and is easy to perform.

[0006] The two patents disclosed above describe typical existing microneedle patches loaded with drugs, with clearly defined drug components. Although existing research has explored loading plant extracts into microneedles, the integration of two different nano-forms of active components from the same plant source into biocompatible hydrogel microneedles, and the use of one component or synergy to trigger responsive drug release, is not reported in the prior art. Summary of the Invention

[0007] Addressing the lack of microneedle patches for anti-inflammatory and analgesic purposes in existing technologies, which primarily consist of exosome vesicles derived from Sichuan pepper and nanoemulsions of Sichuan pepper essential oil, this invention provides an anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper, along with its preparation method and applications.

[0008] This invention provides a novel microneedle patch with excellent biocompatibility and anti-inflammatory and analgesic effects. It combines the natural advantages of plant-derived nanovesicles with the bioactive components of Sichuan pepper essential oil to construct a composite integrated microneedle. This invention uses Sichuan pepper as a raw material to simultaneously prepare Sichuan pepper-derived exosome nanovesicles and Sichuan pepper essential oil nanoemulsions, thus solving the aforementioned technical bottlenecks and achieving efficient utilization of raw materials. Furthermore, the synergistic therapeutic effect of Sichuan pepper-derived exosome nanovesicles and Sichuan pepper essential oil nanoemulsions for anti-inflammatory and analgesic purposes is a first-time discovery and verification.

[0009] Therefore, the present invention aims to provide a novel plant-derived microneedle that integrates highly efficient anti-inflammatory and analgesic effects with intelligent responsive release, so as to achieve the effect of "dual use of the same source and complementary pharmacology", while solving the shortcomings of the existing technology.

[0010] The objective of this invention can be achieved through the following technical solutions: The first aspect of the present invention provides the use of natural Sichuan pepper nanovesicles and natural Sichuan pepper essential oil nanoemulsions in the preparation of anti-inflammatory and analgesic drugs.

[0011] The natural Sichuan pepper nanovesicles are exosome nanovesicles extracted from Sichuan pepper with a particle size of 10-1000 nm, and the natural Sichuan pepper essential oil nanoemulsion has a particle size of 50-500 nm. The two work together to exert anti-inflammatory and analgesic effects by inhibiting multiple signaling pathways. When used in combination as active ingredients in different matrix materials, they have anti-inflammatory and analgesic effects.

[0012] In one embodiment of the present invention, the method for obtaining the natural pepper-derived nanovesicles is as follows: pepper is obtained by cell wall breaking, pulverizing, extracting, gradient centrifuging, and filtering.

[0013] In one embodiment of the present invention, the natural pepper-derived nanovesicles are prepared by first removing dust, washing, drying, breaking down the cell walls and pulverizing, sieving, extracting, gradient centrifuging and filtering the pepper.

[0014] In one embodiment of the present invention, preferably, during gradient centrifugation, the centrifugation is performed first at a low speed, then the speed is gradually increased for centrifugation, and finally ultracentrifugation is performed.

[0015] In one embodiment of the present invention, preferably, the particle size of the natural pepper-derived nanovesicles is 50-200 nm.

[0016] In one embodiment of the present invention, the natural Sichuan pepper essential oil nanoemulsion is prepared from Sichuan pepper waste (including fruit peel residue, etc.) after extracting natural Sichuan pepper source nanovesicles, and the specific method includes: After freeze-drying and pulverizing the waste from Sichuan pepper, Sichuan pepper essential oil was extracted using supercritical carbon dioxide extraction. A natural Sichuan pepper essential oil nanoemulsion is prepared from Sichuan pepper essential oil, surfactant, co-surfactant and water. The natural Sichuan pepper essential oil nanoemulsion is an O / W type nanoemulsion.

[0017] Preferably, the natural Sichuan pepper nanovesicles are extracted from Sichuan pepper from authentic producing areas, with Sichuan Hanyuan green Sichuan pepper being the preferred material; the extraction method of the Sichuan pepper essential oil nanoemulsion is preferably a green and environmentally friendly extraction method, which is prepared by extracting Sichuan pepper waste generated after extracting Sichuan pepper exosome nanovesicles.

[0018] In one embodiment of the present invention, the surfactant is preferably Tween 80; the co-surfactant is preferably 1,2-propanediol; and the preferred mass ratio of the above-mentioned Sichuan pepper essential oil, Tween 80, and 1,2-propanediol is 5-10:3-6:1-3.

[0019] This application uses natural Sichuan pepper nanovesicles and natural Sichuan pepper essential oil nanoemulsions as the main active ingredients to form a dual-active component of Sichuan pepper, so as to achieve the effect of dual use of the same source and complementary pharmacology, and exert a synergistic anti-inflammatory and analgesic effect.

[0020] In a second aspect, the present invention provides an anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper, which is prepared by combining natural Sichuan pepper nanovesicles, natural Sichuan pepper essential oil nanoemulsions and a carrier matrix, wherein the natural Sichuan pepper nanovesicles and natural Sichuan pepper essential oil nanoemulsions are the active ingredients, and the carrier matrix is ​​a polymeric hydrogel material that is biocompatible with the active ingredients.

[0021] In one embodiment of the present invention, the polymer hydrogel material is selected from methacrylamide gelatin (GelMA), gelatin methacrylamide derivatives, methacrylamide hyaluronic acid, alginate photocurable derivatives, etc., with GelMA being a preferred exemplary substrate. The polymer hydrogel material also contains a photoinitiator.

[0022] In one embodiment of the present invention, the final concentration of the natural Sichuan pepper nanovesicles in the microneedle patch is 0.1-1 mg / mL; the final concentration of the natural Sichuan pepper essential oil nanoemulsion in the microneedle patch is 5%-10% (w / v) based on Sichuan pepper essential oil.

[0023] In one embodiment of the present invention, the biocompatible polymer hydrogel material can be selected from types with different strength adaptability, biocompatibility and degradation rate according to the differences of the application object (including but not limited to different skin conditions, different drug administration sites, and different age groups); when GelMA is used as an exemplary hydrogel substrate, the GelMA can be selected as methacrylamide gelatin with an amino substitution degree of 30%-90% according to the differences of the application object.

[0024] In one embodiment of the present invention, when the polymer hydrogel material is selected as GelMA, the mass concentration of GelMA in the carrier matrix is ​​preferably 5%-15%; the mass concentration of the photoinitiator LAP in the carrier matrix is ​​preferably 0.5%-2%; the microneedle patch is cured by irradiation with 365nm ultraviolet light for 15-25min or 405nm blue light for 15-25min.

[0025] In one embodiment of the present invention, the microneedle patch has a square needle array structure. The needle height, needle base diameter, and needle spacing can be reasonably adjusted according to the mold. The puncture force should be greater than 0.3N, the cumulative release rate of the active ingredient within 24 hours should be ≥75%, and the encapsulation rate should remain above 90% after storage at 4°C for 3 months. Due to its tiny array needle structure, it only punctures the surface layer of the skin to form tiny holes, which can heal automatically within a few hours without bleeding or obvious trauma, achieving painless and minimally invasive drug delivery.

[0026] The microneedle patch provided by this invention can alter the arrangement of the lipid layer in the dermis through the components of Sichuan pepper, stimulate local skin temperature to rise by 2-5°C, and increase blood flow by 20%-60%, thereby promoting rapid transdermal drug delivery and release of active components from microneedles.

[0027] The microneedle patch provided by the present invention has a rapid anti-inflammatory and analgesic effect.

[0028] A third aspect of the present invention provides a method for preparing an anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper. The method involves uniformly mixing a carrier matrix with the active ingredients and injecting the mixture into a microneedle mold, followed by vacuum die casting and drying to obtain the microneedle patch.

[0029] Based on the scheme of this application, the cumulative release rate of active components of the microneedle patch is ≥75% within 24 hours, and the encapsulation rate remains ≥90% after storage at 4°C for 3 months.

[0030] A fourth aspect of the present invention provides the application of an anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Zanthoxylum bungeanum, wherein the anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Zanthoxylum bungeanum is used to prepare a transdermal drug delivery formulation with anti-inflammatory and analgesic effects.

[0031] Furthermore, it can be applied to xylene-induced skin inflammation and hot plate test-induced pain responses, achieving rapid and targeted drug release through local application.

[0032] When the anti-inflammatory and analgesic microneedle patch loaded with dual active components from natural Sichuan pepper provided by this invention is used, the active components can be targeted and released slowly in the inflamed area by applying the microneedle patch to the inflamed site. At the same time, the active release effect of Sichuan pepper essential oil nanoemulsion is used to enhance the transdermal drug delivery efficiency and improve bioavailability, thereby effectively exerting anti-inflammatory and analgesic effects. More specifically, the microneedle patch has the following functions: (1) Anti-inflammatory and analgesic: By regulating inflammation-related signaling pathways, inhibiting the expression of pro-inflammatory factors, upregulating the level of anti-inflammatory factors, reducing swelling at the site of inflammation, and reducing pain perception, it can improve the decline in mobility caused by inflammation and pain. (2) Enhanced release and efficacy: By utilizing the properties of Sichuan pepper nanoemulsion to stimulate local skin, promote blood circulation and drug release, solve the problem of low efficiency of passive release in traditional microneedles, and improve the bioavailability of active ingredients at the site of inflammation.

[0033] Specifically, the regulation of inflammatory signaling pathway activity in (1) is manifested in inhibiting the expression of pro-inflammatory factors IL-6 and TNF-α at the site of inflammation, downregulating the phosphorylation level of p-p38 MAPK protein, and promoting the upregulation of anti-inflammatory factor IL-10 at the site of inflammation; the active release effect in (2) can improve drug delivery efficiency by more than 30% compared with traditional passive release.

[0034] This invention provides a clinical application method for an anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper. It should be noted that the core objective of this application is not to provide a specific treatment method; the method described below is merely one alternative. (1) Pretreatment of the affected area: First, gently wipe the inflamed skin with sterile saline or mild medical cleanser to remove hair, oil and dirt from the surface to avoid impurities affecting the microneedle puncture effect; for cases with slight skin damage, use a sterile cotton swab dipped in 75% medical ethanol to disinfect the area around the damage within 0.5cm. Wait for the ethanol to completely evaporate (about 1-2 minutes) before proceeding with subsequent operations to prevent the risk of infection. (2) Application of the patch: Take the microneedle patch and check if the packaging is damaged. Remove the protective film and accurately align the microneedle array surface with the core area of ​​inflammation. Use your fingertip or a sterile pressure pad to gently press the edge and center of the patch for 30 seconds to ensure that the microneedles are fully inserted into the skin surface. For areas with high mobility such as joints, medical breathable tape can be attached to the outside of the patch to help fix it and prevent the patch from shifting due to movement. (3) Maintenance and follow-up treatment: Keep the affected area dry during the application period and avoid prolonged soaking or vigorous rubbing of the patch area. Observe the skin reaction. Slight redness is normal. If itching, redness and swelling worsen, or other discomfort occurs, the patch should be removed immediately. After 2-4 hours of application, the microneedle patch can dissolve on its own. No special bandaging is required for the wound. The tiny pinholes formed can heal on their own within 4-6 hours. Opened or accidentally detached microneedle patches should be disposed of in accordance with medical waste regulations and should not be reused. If a second dose is required, the interval should be more than 12 hours.

[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention uses a highly biocompatible hydrogel material as a carrier substrate (GelMA is an example substrate) and is supplemented with natural Sichuan pepper extract. It not only has excellent biocompatibility, but also has been proven by safety evaluation to have no skin irritation and a cell survival rate of ≥90%.

[0036] 2. The proposed solution utilizes natural Sichuan pepper nanovesicles and natural Sichuan pepper essential oil nanoemulsions, overcoming the inherent drawbacks of poor solubility and low bioavailability of natural products. Natural Sichuan pepper nanovesicles exhibit excellent biocompatibility and stability, enabling effective internalization by mammalian cells and exerting various biological functions such as anti-inflammatory, antioxidant, and tissue regeneration effects. Natural Sichuan pepper essential oil nanoemulsions significantly improve their solubility and transdermal absorption efficiency.

[0037] 3. This invention is the first to integrate two active components derived from the same plant, natural Sichuan pepper nanovesicles and natural Sichuan pepper essential oil nanoemulsions, into microneedles. These two nanocarriers not only improve the bioavailability of various bioactive substances in Sichuan pepper but also simultaneously regulate multiple inflammatory signaling pathways through complementary pharmacological mechanisms, exerting a synergistic effect that is significantly superior to single-component systems. Simultaneously, the Sichuan pepper nanoemulsion can stimulate local warming and promote blood flow, increasing the 24-hour cumulative drug release rate by more than 30%, effectively solving the problem of low passive release efficiency in traditional microneedles. This provides a novel and highly efficient transdermal drug delivery solution for anti-inflammatory and analgesic purposes, particularly suitable for the local treatment of inflammatory pain.

[0038] 4. This invention is the first to extract and simultaneously prepare two active components from Sichuan pepper, resulting in a synergistic effect and complementary pharmacological properties. Simultaneously, this invention significantly improves raw material utilization, reduces resource waste, and aligns with green manufacturing principles. Furthermore, the entire preparation process utilizes mature technologies such as gradient centrifugation, supercritical fluid extraction, and photopolymerization. Optimized key parameters demonstrate high stability, facilitating large-scale production and possessing significant practical application value. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention and to aid in understanding the various technical features and effects, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention (not all embodiments), and therefore should not be considered as a limitation on the scope of protection of the present invention.

[0040] Figure 1 This is a basic characterization diagram of the exosome-like nanovesicles extracted using Sichuan Hanyuan green pepper as an example in this invention. Figure 1 Image A shows the SDS-PAGE protein electrophoresis analysis of Sichuan pepper vesicles and crude Sichuan pepper extract, providing a basis for revealing the protein characteristics of green Sichuan pepper nanovesicles. Figure 1 Pie chart B shows the classification of metabolites from Sichuan pepper vesicles, clearly displaying the proportion of various metabolites such as lipids and lipid analogs, alkaloid derivatives, etc. Figure 1 Image C shows transmission electron microscopy images of the pepper vesicles at different magnifications, demonstrating their typical roundish exosome-like morphology. Figure 1The polydispersity index (PDI) of the dynamic light scattering particle size distribution curve of the pepper vesicles in the middle D is 0.135, reflecting the uniformity of its particle size distribution. Figure 1 The particle size distribution of the nanoparticles in the pepper vesicles in the middle E is statistically analyzed to further verify their particle size range and particle concentration. Figure 1 The middle F is the Zeta potential diagram of the pepper vesicles, showing their surface potential distribution.

[0041] Figure 2 This is a flow chart illustrating the preparation process of Sichuan pepper essential oil nanoemulsion. Figure 2 Figure A is a schematic diagram of the process for extracting Sichuan pepper essential oil from Sichuan pepper waste using supercritical CO2 extraction. Figure 2 Figure B is a schematic diagram of the process for preparing Sichuan pepper essential oil nanoemulsion using a microfluidic-ultrasonic combination method. Figure 2 C represents the positive and negative ion liquid chromatogram of Sichuan pepper essential oil; Figure 2 Figure D shows the main anti-inflammatory and analgesic components and their proportions obtained from mass spectrometry analysis of Sichuan pepper essential oil.

[0042] Figure 3 The preparation process and transdermal drug delivery diagram of anti-inflammatory and analgesic microneedle patches loaded with dual active components from natural Sichuan pepper (taking GelMA and LAP photoinitiators as photocurable substrate materials as examples).

[0043] Figure 4 This is a basic characterization diagram of the anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper in this invention. Figure 4 Image A shows optical photographs of a blank microneedle and a microneedle loaded with Sichuan pepper vesicles and essential oil nanoemulsion, visually demonstrating the differences in appearance between the two types of microneedles. Figure 4 The image in Figure B is a scanning electron microscope image of the drug-loaded microneedle, revealing the basic morphological characteristics of the microneedle from different angles. Figure 4 The middle image (C) is a macroscopic photograph of a mouse after microneedle puncture of the skin, showing the microneedle puncture marks left on the skin surface. Figure 4 In the middle D, the histological HE staining is performed after microneedle puncture of the skin, and the dashed box marks the channel of microneedle puncture; Figure 4 E represents the residual rate curve of microneedles in PBS (pH 7.4, 37℃); Figure 4 Figure F shows the swelling rate curve of microneedles in PBS (pH 7.4, 37℃); Figure 4 G represents the stress-strain curve of the microneedle; Figure 4 The curve of maximum puncture force for the microneedle is represented by H. Figure 4 In Figure I, the fluorescence tracer image of the microneedles shows that the transdermal diffusion of the two fluorescent signals in the skin tissue was observed at 10 min, 30 min, and 60 min, respectively, using red fluorescent labeling of Sichuan pepper essential oil nanoemulsion (Cy5) and green fluorescent labeling of Sichuan pepper vesicles (DiO).

[0044] Figure 5 Figure 1 shows the characterization and mechanism of the promoting effect of Sichuan pepper essential oil nanoemulsion on different transdermal drug delivery models. Figure 5 Image A shows infrared thermal images of the skin surface of rats under different administration methods, including the blank control group, the topical application group, the dressing group, and the microneedle group. Figure 5 Figure B shows ultrasound blood flow imaging test diagrams under different drug administration methods.

[0045] Figure 6 This diagram illustrates the in vitro anti-inflammatory mechanism verification of Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions in this invention. Figure 6 Image A shows a laser confocal image of RAW 264.7 macrophages stained with DCFH-DA fluorescence to detect reactive oxygen species (ROS) levels. Figure 6 In the middle B section, a scatter plot of the inflammatory phenotype of primary chondrocytes from C57 mice detected by flow cytometry (FITC-PI double staining) is shown. Figure 6 C, D, E, and F in the figure represent the band diagrams and quantitative analysis of the levels of phosphorylated nuclear factor-κB (p-NF-κB), cyclooxygenase-2 (COX-2), nuclear nuclear factor E2-related factor 2 (NRF2), and nuclear peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) proteins in primary chondrocytes of C57 mice detected by Western blot.

[0046] Figure 7 for Figure 2 A magnified view of Figure C.

[0047] Figure 8 for Figure 2 A magnified view of the D diagram.

[0048] Figure 9 for Figure 6 Enlarged view of the B-plot (for clarity, the plots corresponding to Control, IL-1β, and Sp-NVs are placed at the top, and the plots corresponding to Sp-VO and Sp-NVs+Sp-VO are placed at the bottom). Detailed Implementation

[0049] To more clearly demonstrate the objectives, technical solutions, and advantages of this invention, the technical solutions are described in detail and comprehensively below. The specific descriptions used herein are merely examples intended to provide a deeper understanding. Unless otherwise stated, the technical and scientific terms generally understood by those skilled in the art have the same meanings.

[0050] It is important to emphasize that the specific terminology used is for illustrative purposes only and does not limit the implementation of the invention. As used herein, unless the context clearly indicates otherwise, the singular form also encompasses the plural form; furthermore, when "comprising" or "including" is used, it means including the stated features, steps, operations, apparatus, components, and combinations thereof. In specific embodiments, if the method steps do not specify conditions, they are generally performed according to conventional methods and conditions in the art, which can be found in detail in authoritative literature or followed as recommended by reagent suppliers and equipment manufacturers.

[0051] The present invention will be specifically described below with reference to embodiments. However, these examples are only for illustrative purposes and should not be considered as limitations on the scope of protection of the present invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0052] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0053] Example 1: Preparation and characterization of Zanthoxylum bungeanum exosome nanovesicles This embodiment uses Sichuan Hanyuan green peppercorns ( Zanthoxylum schinifolium Sieb. et Zucc. Using this as raw material, this variety is a high-quality, authentic medicinal herb with rich and unique bioactive components, providing a high-quality raw material basis for the preparation of nanovesicles with anti-inflammatory and analgesic effects.

[0054] Raw material pretreatment: Fresh green Sichuan peppercorns were taken, and after removing the dust and impurities attached to the surface, they were washed three times with deionized water and drained at room temperature until constant weight. Then, they were pulverized using a traditional Chinese medicine cell wall pulverizer until no obvious whole peppercorn particles were visible, and passed through a 60-mesh sieve. The obtained peppercorn powder was added to an extraction solution containing phosphate buffered saline (PBS) at a material-to-liquid ratio of 1:1.5 (w / v). Each liter of PBS extraction solution contained 5-10 mM Na2HPO4, 130-140 mM NaCl, and 1-5 mM NaH2PO4. The mixture was homogenized three times at 12000 r / min using a high-speed homogenizer, each time for 30 s, with a 1-min interval to avoid heating during homogenization. Subsequently, the mixture was magnetically stirred and extracted at 4℃ for 2 hours. The crude extract of Sichuan peppercorns was obtained by filtration through double-layer nylon gauze.

[0055] Gradient centrifugation and ultracentrifugation extraction method: The pretreated crude extract of Sichuan pepper was subjected to multi-stage differential centrifugation at a low temperature of 4℃ to gradually remove impurities of different particle sizes. First, centrifugation was performed at 300 g for 10 min, and the precipitate was discarded; the supernatant was collected and centrifuged at 3000 g for 15 min, and the precipitate (containing intact cells and tissue fragments) was discarded; then, the supernatant was collected and centrifuged at 10000 g for 15 min, and the precipitate (containing cell nuclei, chloroplasts and other organelles) was discarded; finally, the supernatant was filtered through a 0.8 μm filter membrane and ultracentrifuged at 150000 g for 70 min, and the bottom precipitate was collected, which is the crude extracted Sichuan pepper vesicles.

[0056] Purification and Protein Concentration Quantification: The crude extracted Sichuan pepper vesicles were resuspended in PBS and then filtered sequentially through 0.45 μm and 0.22 μm filters to remove residual large particles and potential bacteria. The purified Sichuan pepper vesicle solution was aliquoted into sterile cryovials and stored at -80°C for later use. The protein content of the Sichuan pepper vesicles was determined using the BCA protein quantification kit from Beyotime Biotechnology Co., Ltd. to characterize the relative amount of vesicles used in subsequent experiments. Standard protein solutions of different concentrations were prepared according to the kit instructions, and the absorbance values ​​of each concentration were measured. Linear regression analysis was performed on absorbance y versus protein concentration x, and a standard curve was plotted. The results showed a good linear relationship in the range of 0–3.0 μg / μL (R²). 2 ≥0.996).

[0057] 5 μL of purified Sichuan pepper vesicle solution was processed according to the kit procedure, and the protein concentration was calculated to be 1.38 ± 0.15 μg / μL by substituting the values ​​into the standard curve. Subsequently, the protein expression characteristics of Sichuan pepper vesicles were analyzed by SDS-PAGE electrophoresis. 20 μg of Sichuan pepper vesicles and 20 μL of crude Sichuan pepper extract were mixed with loading buffer, denatured in a metal bath for 5 minutes, loaded, and subjected to 12% separating gel electrophoresis. After staining with Coomassie Brilliant Blue and destaining, the results were observed. Figure 1 As shown in Figure B, the molecular weight range of proteins is indicated by the Marker (kDa 180-15). By comparing the differences in protein composition between the two, the molecular weight of proteins in the Sichuan pepper vesicles is mainly distributed between 15~40 kDa and 70~130 kDa, showing 5 characteristic protein bands, reflecting specific protein composition characteristics and their correlation with the protein composition of crude Sichuan pepper extract.

[0058] High-resolution untargeted metabolomics analysis: Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was used to comprehensively analyze the composition of metabolites in *Zanthoxylum bungeanum* vesicles. The *Zanthoxylum bungeanum* vesicle solution stored at -80℃ was slowly thawed at 4℃. Then, 100 μL of the sample was weighed and added to 400 μL of pre-cooled methanol-acetonitrile-water (2:2:1, v / v) mixture, and vortexed for 1 minute to ensure thorough mixing. The sample was then subjected to ultrasonic extraction at 4℃ and 150W for 30 minutes. After extraction, the mixture was allowed to stand at -20℃ for 10 minutes to promote protein precipitation. Next, the sample was centrifuged at 4℃ and 15000 g for 15 min. The supernatant was collected, transferred to a new centrifuge tube, and dried to constant weight using a vacuum freeze dryer. Before mass spectrometry analysis, 100 μL of a 1:1 (v / v) acetonitrile-water solution was added to reconstitute the sample residue. After vortexing for 2 minutes, the sample was centrifuged at 4°C and 15000 g for 15 minutes. The supernatant was collected, filtered through a 0.22 μm organic phase filter, and then placed in a sample vial for analysis. This test was commissioned to Hangzhou Lianchuan Biotechnology Co., Ltd. for testing. The analysis was performed using an Agilent 1290 Infinity II ultra-high performance liquid chromatograph and an AB Sciex Triple TOF 6600 quadrupole time-of-flight mass spectrometer. Metabolite data were acquired in both positive and negative ion modes and analyzed qualitatively and quantitatively using a database. The specific results are presented in […]. Figure 1 B in the middle.

[0059] Particle size and morphological characterization: The morphology and structure were observed by transmission electron microscopy (TEM). 20 μL of *Zanthoxylum bungeanum* vesicle solution was diluted 5-fold with sterile PBS, added to a copper grid, and allowed to air dry at room temperature for 10 min. Then, 2% phosphotungstic acid solution (pH 7.0) was added for negative staining for 3 min. Excess stain was absorbed with filter paper, and the solution was allowed to air dry again. The solution was then observed and photographed under TEM. The results are as follows: Figure 1 As shown in Figure C, the vesicles exhibit a typical round or teacup-shaped exosome-like morphology, with a clear phospholipid bilayer structure, and are fully formed and plump, consistent with the classic characteristics of plant-derived exosome-like nanovesicles. Dynamic light scattering (DLS) combined with nanoparticle tracking analysis (NTA) was used to determine the particle size distribution and zeta potential of the purified *Zanthoxylum bungeanum* vesicles. The diluted *Zanthoxylum bungeanum* vesicle solution was added to a special glass test dish for testing. The results are as follows... Figure 1 As shown in Figures D and F, the average particle size of the *Zanthoxylum bungeanum* vesicles is 138.6 nm, with a particle size distribution concentrated in the range of 30-300 nm, consistent with the typical particle size characteristics of plant-derived exosome-like nanovesicles; Figure 1The E and Zeta potential detection results showed that its surface was negatively charged, with an absolute potential value between 15 and 20 mV, which is consistent with the charged properties of plant-derived nanovesicles, indicating that the vesicle solution has good colloidal stability.

[0060] Example 2: Preparation and basic characterization of Sichuan pepper essential oil nanoemulsion In this embodiment, to achieve efficient utilization of Sichuan pepper resources, Sichuan pepper essential oil is prepared using waste materials such as Sichuan pepper peel residue after extracting Sichuan pepper vesicles in Example 1 as raw materials, constructing a green preparation system with "one material for two uses" and realizing an environmentally friendly process of "turning waste into treasure".

[0061] Extraction of Sichuan pepper essential oil: such as Figure 2 As shown in Figure A, the waste Sichuan pepper was pre-frozen at -80℃ for 2 hours, then freeze-dried in a freeze dryer for 24 hours. Afterward, it was pulverized using a high-speed grinder until no obvious intact particles remained, and then the powder was collected through an 80-mesh sieve. Supercritical carbon dioxide extraction was used to extract Sichuan pepper essential oil. Compared to traditional solvent extraction or steam distillation, this method effectively preserves heat-sensitive active ingredients and leaves no solvent residue. The extraction parameters were set as follows: extraction pressure 30 MPa, extraction temperature 40℃, CO2 flow rate 20 L / h. Static extraction was performed for 2 hours, followed by dynamic extraction for 1 hour. The extracted product was collected as Sichuan pepper essential oil and stored in a sealed container at 4℃ for later use.

[0062] Preparation of Sichuan pepper essential oil nanoemulsion: such as Figure 2In step B, the pepper essential oil prepared in step (1) is used as the oil phase, Tween 80, a food-grade nonionic surfactant, is used as the emulsifier, 1,2-propanediol is used as the co-surfactant, and deionized water is used as the aqueous phase. The phase system is configured according to the ratio of oil phase: emulsifier: co-surfactant: deionized water = 25:15:5:55 (w / w / w). The synergistic effect of Tween 80 and 1,2-propanediol can further reduce the interfacial tension between oil and water, improve the stability of the emulsion system, and provide a guarantee for the stable formation of O / W type nanoemulsions. A glass chip microfluidic device with a microchannel size of 50 μm is selected. The oil phase and the mixed aqueous solution containing Tween 80 and 1,2-propanediol (the emulsifier and co-surfactant are pre-dissolved in deionized water) are injected into the two injection channels of the device respectively. The flow rate of the oil phase is set to 50 μL / min and the flow rate of the aqueous phase is set to 250 μL / min. A laminar flow state is formed in the microchannel to achieve uniform mixing of the oil phase and the aqueous phase, resulting in a clear and transparent pre-emulsion. This process avoids localized imbalances caused by traditional stirring, laying a foundation for uniformity in subsequent droplet refinement. The microfluidically prepared pre-emulsion is then directly introduced into the ultrasonic processing unit. A probe-type ultrasonic instrument is used, with an ultrasonic power of 150W, to ultrasonically emulsify for 15 minutes. During ultrasonication, the system temperature is maintained at ≤25℃ using an ice-water bath until a clear, homogeneous, and non-layered Sichuan pepper essential oil nanoemulsion is obtained. This nanoemulsion is then stored at 4℃ in the dark for characterization. Alternatively, this step can be prepared using a microfluidic high-pressure homogenizer. The mixture is directly transferred to the high-pressure homogenizer, dispersed using a high-speed disperser at 10000 r / min, and then processed three times using a microfluidic high-pressure homogenizer to obtain the Sichuan pepper essential oil nanoemulsion.

[0063] Basic characterization of Sichuan pepper essential oil: High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to qualitatively identify and quantitatively analyze the characteristic active components of Sichuan pepper essential oil in nanoemulsions, verifying the retention effect of the process on active components. Samples were slowly thawed at 4℃, and an appropriate volume (0.5-1 mL; for larger sample volumes, freeze-drying concentration can be performed) was accurately measured into centrifuge tubes. Twice the volume of extraction buffer (methanol / acetonitrile, 1:1, v / v) was added, vortexed for 60 s, and subjected to low-temperature ultrasonic extraction for 30 min. The mixture was then centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was collected and placed at -20℃ for 1 h to precipitate proteins. Then, it was centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was vacuum dried, reconstituted with 100 μL of 30% acetonitrile solution, vortexed, and centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was then used for instrumental analysis. Figure 2 C, Figure 7As shown, in positive ion mode, characteristic salicornin components such as hydroxyα-salicornin, hydroxyγ-salicornin, and β-salicornin, as well as active substances such as gabapentinolactam and docosahexaenoic acid acetamide, were identified; in negative ion mode, components with anti-inflammatory activity such as cinnamic acid, ferulic acid, azelaic acid, and capsaicin were identified.

[0064] Combined with relative area percentage analysis, such as Figure 2 D, Figure 8 As shown, hydroxy-γ-sanshool had the highest relative content (10.115%), followed by oleuropein (2.697%), perillol (2.487%), and β-sanshool (1.606%). These components are the core material basis for the analgesic and anti-inflammatory effects of Sichuan pepper, and their retention rate in nanoemulsions is relatively high, confirming that the microfluidic-ultrasound combined process can efficiently retain the active ingredients of Sichuan pepper essential oil. This testing was commissioned to Beijing Zhongke Baice Information Technology Co., Ltd., and the data acquisition instrument system mainly included ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometry (Q Exactive HFX, Thermo, USA).

[0065] Example 3: Preparation of GelMA photocurable composite hydrogel microneedle patch loaded with natural Sichuan pepper extract. In this embodiment, the Sichuan pepper nanovesicles prepared in Example 1 and the Sichuan pepper essential oil nanoemulsion prepared in Example 2 are integrated into a GelMA matrix, and a composite microneedle patch is prepared by photocuring technology to achieve synergistic loading of the two active components. All the following steps are prepared using a mouse transdermal microneedle formulation.

[0066] Preparation of GelMA precursor solution: Weigh methacrylamide gelatin (GelMA 60, amino substitution degree 60±5%, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.), add 1×PBS solution in a sterile laminar flow hood, and then place the prepared GelMA solution in a preheated water bath at 60℃ and heat continuously until completely dissolved to obtain a 15% (w / v) GelMA solution. Using the Sichuan pepper nanovesicles from Example 1, select a concentration of 0.8 mg / mL and the Sichuan pepper essential oil nanoemulsion from Example 2, and slowly add them dropwise at a concentration of 15% (w / v) to the GelMA solution, stirring magnetically at 500 r / min for 10 min until uniformly mixed. Finally, add the photoinitiator LAP (purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) to a final concentration of 0.3% (w / v), and continue stirring for 5 min. The entire operation should be carried out under light-shielding conditions with aluminum foil to avoid premature exposure of LAP to light.

[0067] Microneedle molding and photopolymerization: such as Figure 3As shown, a PDMS microneedle mold (model EFL-MMN-600, Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) was sterilized with 75% ethanol for 30 min, then rinsed three times with deionized water and air-dried in a clean bench. The mold is a square patch structure containing a 20×20 microneedle array with a needle spacing of 550 μm, a needle base diameter of 250 μm, and a needle height of 600 μm. The aforementioned GelMA precursor solution was injected into the mold and placed in a vacuum drying oven at ≤0.1 MPa for 10 min to degas, ensuring the solution completely filled the micropores of the mold without any residual air bubbles. The mold was then dried in a room temperature drying oven for 48 h until the edges were visibly dry. The dried microneedle mold was then placed under a 365 nm UV LED light source at 25 mW / cm². 2 Irradiate the microneedles with light at an intensity of 30-40 seconds to initiate cross-linking and curing of the GelMA. The light irradiation parameters have been optimized through preliminary experiments to ensure sufficient curing of the microneedles while preventing the active ingredients from being deactivated due to excessive light exposure. Peel the cured microneedle patch from the mold and check the integrity of the microneedles. Place the qualified microneedle patches in a sealed bag with desiccant for storage.

[0068] Example 4: Basic Characterization of Microneedles This embodiment performs basic characterization on composite microneedles loaded with Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions, and systematically analyzes their morphology, skin puncture performance, degradation and swelling characteristics, mechanical properties and transdermal delivery effect, providing a basis for carrier performance in subsequent pharmacodynamic studies.

[0069] The drug-loaded microneedles used in this embodiment were all prepared using the preparation method mentioned in Example 3. The preparation method for blank microneedles or single-component microneedles was the same as that for composite microneedles, except that an equal volume of PBS was used instead of the active ingredient mixture.

[0070] The specific methods, intended purposes, and references for each characterization item are as follows: Morphological characteristics: such as Figure 4 In section A, composite drug-loaded microneedles and blank microneedles were taken and their macroscopic morphology was photographed using an optical camera. The blank microneedles were transparent, while the composite drug-loaded microneedles, due to the loading of the Sichuan pepper-derived component, exhibited a characteristic pale yellow color, preliminarily indicating that the active ingredient had been successfully loaded. Scanning electron microscopy (SEM) was used to observe the microneedles. Before detection, the microneedle samples were sputtered with gold to enhance conductivity. Subsequently, the morphology of the microneedle array and individual needles was photographed under an accelerating voltage of 5 kV. Figure 4 As shown in Figure B, the intended purpose of this characterization is to determine whether the microneedle array is complete and undamaged, whether the needle tip is sharp, whether the needle surface is smooth, and whether a porous structure conducive to drug release is formed inside. The relevant detection methods refer to the existing standard procedures for microneedle morphology characterization.

[0071] Skin puncture capability verification: After hair removal and disinfection of the backs of adult BALB / c mice, microneedle patches were applied to the skin surface and gently pressed for 15 seconds before removal. Figure 4 In the middle stage, the skin puncture marks were photographed and observed to determine the uniformity of the punctures; simultaneously, full-thickness skin of mice was cut, fixed with 4% paraformaldehyde, and used to prepare paraffin tissue sections. Subsequently, histological HE staining was used to observe the effective puncture depth of the microneedles in the stratum corneum. Figure 4 As shown in Figure D, the drug-loaded microneedles prepared using the above method can effectively penetrate the epidermis and reach the dermis.

[0072] Evaluation of Degradation and Swelling Characteristics: The swelling and degradation characteristics of microneedles in a simulated physiological environment were studied using the immersion-weighing method. Microneedle samples were accurately weighed (denoted as initial mass m0) and placed in PBS buffer (pH 7.4) at a constant temperature of 37℃. Samples were removed after immersion for 1, 2, 4, 8, 12, and 24 h, and after the surface moisture was absorbed with absorbent paper, they were quickly weighed (denoted as m). t ), calculate the swelling ratio at different time points (swelling ratio = (m t -m0) / m0×100%); then continue to remove on days 3, 5, 7, and 14, absorb the surface moisture, weigh, and calculate the residual rate (residual rate = m t / m0×100%). For example Figure 4 As shown in E and F, this analysis aims to clarify whether the swelling kinetics of microneedles are stable and without burst release, providing a basis for subsequent drug release characteristic studies.

[0073] Mechanical performance testing: The mechanical properties of the microneedles were tested using a universal testing machine. First, the microneedle patch was fixed on a compression table with the tip facing upwards. Then, a single microneedle was axially compressed at a rate of 0.5 mm / min until it broke and rebounded. The pressure change curves during the process were recorded. Figure 4 The presence of G indicates that the loading of active ingredients from Sichuan pepper did not compromise the mechanical properties of the microneedle matrix. Figure 4 The curve in Figure H represents the maximum puncture force of the microneedle. The maximum puncture force of the drug-loaded microneedle is >0.5N, and the initial bending puncture force is >2.5N, which meets the mechanical requirements for minimally invasive skin puncture. The core purpose of this test is to ensure that the microneedle has sufficient compressive strength and breaking force to meet the puncture requirements when administering drugs through the skin surface.

[0074] Transdermal drug delivery tracking validation: Sichuan pepper essential oil nanoemulsions were labeled with Cy5 red fluorescent dye, and Sichuan pepper nanovesicles were labeled with DiO green fluorescent dye to prepare fluorescently labeled drug-loaded composite microneedles. Microneedle patches were then attached to the hairless areas of the backs of mice. After 10, 30, and 60 minutes, the microneedle patches were removed, and the mice were sacrificed. Skin tissue from the microneedle administration sites was collected. Skin sections were prepared from the microneedle penetration sites, and the signal distribution corresponding to the drug was observed using a fluorescence microscope (excitation wavelengths: Cy5 633nm, DiO 488nm). The scanned images were superimposed and observed in conjunction with bright-field microscopy. Figure 4 As shown in Figure I, red (Sichuan pepper essential oil nanoemulsion) and green (Sichuan pepper vesicles) fluorescent signals were observed in the dermis layer of the skin after 10 minutes. After 30 minutes, the fluorescent signals gradually diffused into the muscle layer, and after 60 minutes, the fluorescence signals clearly penetrated the connective tissue. This method also indirectly verifies that the active ingredients in Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions have been effectively loaded into the GelMA matrix.

[0075] Example 5: Characterization of the promoting effect of Sichuan pepper essential oil nanoemulsion on transdermal drug delivery This embodiment aims to explore the promoting effect and corresponding potential mechanism of the Sichuan pepper essential oil nanoemulsion mentioned in this invention on transdermal drug delivery via composite microneedles. Local thermal effects and hemodynamic changes after loading Sichuan pepper essential oil nanoemulsion into different drug delivery models were analyzed using infrared thermography and ultra-high frequency small animal ultrasound imaging techniques. The Sichuan pepper essential oil nanoemulsion can gently stimulate the local skin to produce a temperature rise effect, thereby promoting subcutaneous microvascular dilation and accelerating local blood circulation. The increased blood flow not only helps to accelerate the dissolution of the GelMA hydrogel matrix but also promotes the efficient release and absorption of active ingredients, forming an intelligent, self-triggered responsive drug release system. This mechanism is consistent with the reported temperature-responsive microneedle design concept, but its triggering originates from the drug itself rather than external stimulation, offering greater application convenience.

[0076] First, GelMA microneedle patches loaded with Sichuan pepper essential oil nanoemulsion were prepared using the method described in Example 3. Then, control groups were set up using direct application and cloth immersion methods, with the same Sichuan pepper essential oil nanoemulsion loading dose, to further clarify the advantages of microneedles in promoting transdermal absorption. The specific implementation method is as follows: Infrared thermometry: SPF-grade adult SD rats were selected, and the backs of the rats were shaved. The shaved area was divided into four equal-area square regions (each 1cm × 1cm), serving as the blank control group, direct application group, dressing group, and microneedle drug delivery group, respectively. A non-contact infrared thermal imager (model: FLIR T620, temperature measurement range set to 25-40℃) was used to monitor the temperature changes of each area and surrounding skin in real time after drug administration, and thermal images were acquired at each time point. Figure 3 The average value was taken and statistically analyzed to assess the differences in local thermal effects after transdermal drug delivery under different administration methods. For example... Figure 5 As shown in Figure A, 5 minutes after drug administration, the temperatures at the drug administration sites in each group were 33.4℃, 35.2℃, 36.5℃, and 38.8℃, respectively. This indicates that the temperature increase at the drug administration site was most significant in the microneedle group, suggesting that the local metabolism or blood flow activity is enhanced after the Sichuan pepper essential oil nanoemulsion is delivered transdermally via microneedles, resulting in better transdermal absorption.

[0077] Ultrasonic blood flow observation: A high-frequency small animal ultrasound imaging system (Shenzhen Huanying Company, model: Sonorover UR820) was used to perform blood flow imaging and flow velocity measurement on the subcutaneous tissue vessels in the application area under different drug administration modes. By comparing the differences before and after drug administration and with the control group, the study verified whether Sichuan pepper essential oil nanoemulsion could significantly increase local blood flow and velocity. Figure 5 As shown in Figure B, the blood flow velocity in the blank control group was 277.31 mm / s; it increased to 293.15 mm / s in the topical application group, 340.69 mm / s in the dressing group, and significantly increased to 393.51 mm / s in the microneedle group. The increased blood flow velocity indicates that, within the same time frame, microneedles can better improve local hemodynamics, further accelerating drug distribution and efficacy, demonstrating the significant advantages of microneedles in promoting transdermal drug delivery.

[0078] Example 6: Validation of the in vitro anti-inflammatory mechanism of Sichuan pepper vesicle exosomes and essential oil nanoemulsions This embodiment aims to explore the anti-inflammatory effects and mechanisms of the combined in vitro application of Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions in this invention. By analyzing the effects of different treatment groups on intracellular oxidative stress, inflammatory signaling pathways, and antioxidant factors, the aim is to preliminarily reveal its potential mechanism of action and provide some theoretical support for subsequent animal model experiments.

[0079] First, Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions were prepared using the methods described in Examples 1 and 2, respectively. RAW 264.7 macrophages and C57 mouse chondrocytes were selected as in vitro models to verify the effects of reagent treatment on relevant intracellular biomarkers. The specific experimental methods are as follows: Oxidative stress assay: Transwell chambers were placed in 6-well plates, and RAW 264.7 cells (density 5 × 10⁶) were seeded in the lower chamber. 4Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. After cell attachment, they were randomly divided into 6 groups: ① Normal control group (NC); ② TNF-α induction group; ③ Sichuan pepper vesicle group; ④ Sichuan pepper essential oil nanoemulsion group; ⑤ Combined drug administration group; ⑥ Composite microneedle group (microneedles placed tip-down on top of the matrix gel in the upper chamber of the Transwell). Except for the NC group, all other groups were stimulated with 20 ng / mL TNF-α. After 24 hours, DCFH-DA probe staining was performed, and ROS level changes were analyzed using laser confocal microscopy. Figure 6 As shown in Figure A, the combined treatment group and the microneedle group significantly reduced intracellular ROS levels, and compared with other treatment groups, the effect of clearing oxidative stress was faster and better.

[0080] Apoptosis analysis: SPF-grade C57 mouse primary chondrocytes were extracted and cultured in vitro. When the cells reached 70-80% confluence, they were divided into three groups: untreated group, IL-1β-induced group, and groups treated alone or in combination. Changes in inflammatory phenotypes after treatment were analyzed using FITC-PI double staining combined with flow cytometry and FlowJo software. Figure 6 B, Figure 9 The combined treatment group showed that it significantly improved the abnormal cell phenotype and apoptosis induced by IL-1β.

[0081] Inflammation and antioxidant factor detection: Western blot was used to detect the expression of p-NF-κB and COX-2, as well as the changes in the levels of NRF2 and PGC-1α in primary chondrocytes of C57 mice. Figure 6 As shown in C~F, both Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions exhibited effective regulation of inflammatory signaling pathways, and also showed potential synergistic anti-inflammatory advantages.

[0082] Example 7: Anti-inflammatory and analgesic pharmacodynamic study of microneedles loaded with Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions This embodiment aims to evaluate the anti-inflammatory and analgesic effects of Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions. Sichuan pepper vesicles were extracted and prepared using the method described in Example 1; the Sichuan pepper essential oil nanoemulsion was prepared using the same batch of Sichuan pepper waste materials as described in Example 2. Pharmacodynamic validation was performed using two classic animal models of inflammation and pain, and the preparation and application methods of the composite microneedles were clarified.

[0083] The following experimental animals were all SPF-grade C57 mice, weighing 20-24g, with half males and half females. After acclimatization, the mice were randomly divided into 6 groups according to their weight, with more than 6 mice in each group. The specific groups are as follows: ① Blank control group; ② Model control group: only modeling treatment was performed; ③ Positive drug control group: after modeling, flurbiprofen gel cream (each patch contains 40mg flurbiprofen) was applied topically to the affected area; ④ Low-dose composite microneedle group: each microneedle patch contained 50μg of Sichuan pepper vesicles and 5mg of Sichuan pepper essential oil nanoemulsion; ⑤ Medium-dose composite microneedle group: each microneedle patch contained 100μg of Sichuan pepper vesicles and 10mg of Sichuan pepper essential oil nanoemulsion; ⑥ High-dose composite microneedle group: each microneedle patch contained 200μg of Sichuan pepper vesicles and 20mg of Sichuan pepper essential oil nanoemulsion. The administration method was standardized as follows: Mice were treated with depilatory cream and then disinfected with 75% ethanol at the administration site. Microneedles were then applied to the depilated area and secured with medical tape. In the positive control group, identical square patches of the same size as the microneedles were cut and applied topically. The administration frequency and modeling time were the same as in the composite microneedle group. Specific implementation methods and efficacy results are as follows: Xylene-induced mouse ear swelling and inflammation model experiment: Xylene and ether were mixed at a ratio of 1:3 (v / v) to prepare an inflammatory agent. 50 μL was drawn using a microsyringe and evenly applied to both the anterior and posterior surfaces of the right ear of mice. The left ear was left untreated as a blank control. Within 30 minutes of model establishment, the mice were administered the drug according to the above grouping. After 4 hours of observation, the mice were euthanized by cervical dislocation. Ear flaps were punched at the same location on both ears using an 8 mm diameter punch. The flaps were weighed on an electronic analytical balance, and the degree of ear swelling and the swelling inhibition rate were calculated. Ear swelling degree = weight of right ear flap - weight of left ear flap; swelling inhibition rate = (average swelling degree of the model control group - average swelling degree of the drug-treated group) / average swelling degree of the model control group × 100%. The results are shown in the table below: Mouse hot plate analgesia model experiment: Before the experiment, the hot plate apparatus was preheated to 55℃±0.5℃ and kept stable. Mice with a baseline pain threshold of 5-30 seconds were selected (pain threshold determination criteria: the time it takes for a mouse to lick its hind paw, lift its paw to avoid the heat, or jump after being placed on the hot plate is the pain threshold; if no response is observed after 60 seconds, it is counted as 60 seconds to avoid scalding the mice). The mice were administered the drug according to the aforementioned grouping protocol. The pain threshold of each group of mice was measured before administration (0 min) and at 30 min and 60 min after administration. The maximum analgesia rate was calculated as follows: Maximum analgesia rate = (maximum pain threshold after administration - baseline pain threshold before administration) / (60 - baseline pain threshold before administration) × 100%. Mice exhibiting abnormal paw licking were excluded before the experiment. Specific experimental results are as follows: In summary, this embodiment, using a xylene-induced acute inflammation model of mouse ear swelling combined with a temperature-sensitive pain model represented by the hot plate model, systematically validated through multi-dimensional analysis that transdermal administration of composite microneedles loaded with Sichuan pepper vesicles and Sichuan pepper essential oil nanoemulsions significantly inhibited the occurrence and development of acute inflammation while alleviating temperature-sensitive pain. The high-dose group showed anti-inflammatory and analgesic effects comparable to the positive control drug, with stable and sustained efficacy. The multi-component and multi-target advantages of Sichuan pepper natural extracts hold promise for achieving broader-spectrum anti-inflammatory and analgesic effects. Its all-plant-derived nature and the reuse of Sichuan pepper waste also contribute to its good biosafety and environmental friendliness, providing a valuable example and broad application prospects for developing more natural product-based intelligent transdermal drug delivery systems.

[0084] The technical solution of this application has been described in detail through the above embodiments, but this patent is not limited to the description of these specific embodiments. Those skilled in the art should understand that any improvements to the present invention, or equivalent substitutions of raw materials, addition of auxiliary components, and changes in the selection of specific embodiments, etc., all fall within the protection and disclosure scope of this patent.

[0085] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of natural Sichuan pepper nanovesicles and natural Sichuan pepper essential oil nanoemulsions in the preparation of anti-inflammatory and analgesic drugs, characterized in that, in, The natural Sichuan pepper nanovesicles are exosome nanovesicles extracted from Sichuan pepper with a particle size of 10-1000 nm, and the natural Sichuan pepper essential oil nanoemulsion has a particle size of 50-500 nm. The method for obtaining the natural Sichuan pepper nanovesicles is as follows: Sichuan pepper is obtained by cell wall breaking, pulverizing, extracting, gradient centrifugation, and filtration. The natural Sichuan pepper essential oil nanoemulsion is prepared from Sichuan pepper waste material after extracting natural Sichuan pepper nanovesicles. The specific method includes: After freeze-drying and pulverizing the waste from Sichuan pepper, Sichuan pepper essential oil was extracted using supercritical carbon dioxide extraction. A natural Sichuan pepper essential oil nanoemulsion is prepared from Sichuan pepper essential oil, surfactant, co-surfactant and water. The natural Sichuan pepper essential oil nanoemulsion is an O / W type nanoemulsion.

2. The application of the natural Sichuan pepper-derived nanovesicles and natural Sichuan pepper essential oil nanoemulsions according to claim 1 in the preparation of anti-inflammatory and analgesic drugs, characterized in that, The particle size of the natural Sichuan pepper-derived nanovesicles is 50-200 nm.

3. The application of the natural Sichuan pepper-derived nanovesicles and natural Sichuan pepper essential oil nanoemulsions according to claim 1 in the preparation of anti-inflammatory and analgesic drugs, characterized in that, The surfactant is Tween 80; the co-surfactant is 1,2-propanediol; the mass ratio of the above-mentioned Sichuan pepper essential oil, Tween 80, and 1,2-propanediol is 5-10:3-6:1-3.

4. A microneedle patch for anti-inflammatory and analgesic purposes, loaded with dual active components derived from natural Sichuan pepper, characterized in that... It is prepared by combining natural Sichuan pepper nanovesicles, natural Sichuan pepper essential oil nanoemulsions and a carrier matrix. Among them, natural Sichuan pepper nanovesicles and natural Sichuan pepper essential oil nanoemulsions are active ingredients, and the carrier matrix is ​​a high molecular hydrogel material that is biocompatible with the active ingredients. The natural Sichuan pepper nanovesicles are exosome nanovesicles extracted from Sichuan pepper with a particle size of 10-1000 nm, and the natural Sichuan pepper essential oil nanoemulsion has a particle size of 50-500 nm. The method for obtaining the natural Sichuan pepper nanovesicles is as follows: Sichuan pepper is obtained by cell wall breaking, pulverizing, extracting, gradient centrifugation, and filtration. The natural Sichuan pepper essential oil nanoemulsion is prepared from Sichuan pepper waste material after extracting natural Sichuan pepper nanovesicles. The specific method includes: After freeze-drying and pulverizing the waste from Sichuan pepper, Sichuan pepper essential oil was extracted using supercritical carbon dioxide extraction. A natural Sichuan pepper essential oil nanoemulsion is prepared from Sichuan pepper essential oil, surfactant, co-surfactant and water. The natural Sichuan pepper essential oil nanoemulsion is an O / W type nanoemulsion.

5. The anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper according to claim 4, characterized in that, The polymer hydrogel material is selected from methacrylamide gelatin, gelatin methacrylamide derivatives, methacrylamide hyaluronic acid, and alginate photocurable derivatives.

6. The anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper according to claim 4, characterized in that, The final concentration of the natural Sichuan pepper nanovesicles in the microneedle patch is 0.1-1 mg / mL; the final concentration of the natural Sichuan pepper essential oil nanoemulsion in the microneedle patch is 5%-10% (w / v) based on Sichuan pepper essential oil.

7. The anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper according to claim 4, characterized in that, When the polymer hydrogel material is GelMA, the mass concentration of GelMA in the carrier matrix is ​​5%-15%; the mass concentration of the photoinitiator LAP in the carrier matrix is ​​0.5%-2%; the microneedle patch is cured by irradiation with 365nm ultraviolet light for 15-25 minutes or irradiation with 405nm blue light for 15-25 minutes.

8. The anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper according to claim 4, characterized in that, The microneedle patch has a square needle array structure, a puncture force greater than 0.3N, an active component cumulative release rate of ≥75% within 24 hours, and an encapsulation rate of over 90% after storage at 4℃ for 3 months.

9. The method for preparing the anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper as described in claim 4, characterized in that, After the carrier matrix and active ingredients are mixed evenly, they are injected into a microneedle mold, and then dried and demolded through vacuum die casting to obtain a microneedle patch.

10. The application of the anti-inflammatory and analgesic microneedle patch loaded with dual active components derived from natural Sichuan pepper as described in claim 4, characterized in that, The anti-inflammatory and analgesic microneedle patch loaded with dual active components from natural Sichuan pepper is used to prepare transdermal drug delivery formulations with anti-inflammatory and analgesic effects.

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