Active factor spray for skin inflammatory and allergic pruritus

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses an active factor spray for skin inflammatory and allergic pruritus. The composition is prepared from hyaluronic acid, lycorine N-demethylated derivative, hedyotis diffusa polysaccharide, purified water and exosome hybrid peptide according to a specific ratio. Wherein the exosome hybrid peptide is composed of plant exosome loaded blue copper peptide, palmitoyl pentapeptide-4 and FGF2 growth peptide, and an efficient targeted delivery system is formed. Through combination of multi-component compounding and a delivery technology, the anti-inflammatory, itching-relieving and skin barrier repairing effects are synergistically achieved. And a safer and more comprehensive non-hormone local treatment new choice is provided for the skin inflammatory and allergic pruritus.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to an active factor spray for inflammatory and allergic pruritus of the skin. Background Technology

[0002] Pruritus is one of the most common clinical symptoms of various inflammatory and allergic diseases, such as atopic dermatitis, contact dermatitis, eczema, and urticaria. Its pathogenesis is complex, involving the release of various mediators such as histamine, neuropeptides, and cytokines, as well as neurosensitization processes. Long-term or recurrent itching not only seriously affects the patient's quality of life, but can also further damage the skin barrier due to scratching, aggravating the inflammatory response and forming a vicious cycle of "itching-scratching".

[0003] Currently, commonly used topical antipruritic medications in clinical practice mainly include glucocorticoids, calcineurin inhibitors, antihistamines, and local anesthetics. Although these medications can relieve symptoms to some extent, their application still has significant limitations: long-term use of glucocorticoids can cause adverse reactions such as skin atrophy and telangiectasia; calcineurin inhibitors may have potential long-term safety concerns; oral antihistamines are often accompanied by central nervous system side effects such as drowsiness and fatigue; and traditional topical antipruritic preparations often only act on the surface, failing to adequately regulate deep inflammation and neurosensitization, and have limited effectiveness in repairing the skin barrier function.

[0004] In recent years, although some studies have attempted to use bioactive peptides (such as copper peptide and growth factors) or plant extracts for skin repair, these ingredients, when used alone for topical application, suffer from problems such as low transdermal absorption, easy inactivation in the skin environment, and difficulty in targeted delivery to the desired action level. While exosomes have attracted attention as novel delivery carriers, there are currently no reports of using them to synergistically load multiple functional peptides (such as copper peptide, palmitoyl pentapeptide-4, and FGF2 growth peptide) and combine them with lycorine derivatives, Hedyotis diffusa polysaccharides, and other ingredients to construct a multi-target, multi-mechanism synergistic 'antipruritic-anti-inflammatory-repair' integrated topical spray formulation.

[0005] Furthermore, the specific anti-inflammatory and neuromodulatory activities of lycorine N-demethylated derivatives, as well as the scientific formulation of them with immunomodulatory polysaccharides from Hedyotis diffusa and highly moisturizing hyaluronic acid, and the physical integration with specific functional peptides through exosome technology (such as specific plant exosomes) to construct a multi-target, multi-mechanism synergistic "antipruritic-anti-inflammatory-repair" integrated local spray formulation, are still a blank in existing technologies. Summary of the Invention

[0006] To address the problems of significant drug side effects, limited repair efficacy, and low delivery efficiency of active ingredients in existing treatments for inflammatory and allergic pruritus, this application proposes an active factor spray for inflammatory and allergic pruritus. It combines hyaluronic acid, lycorine N-demethylated derivatives, and Hedyotis diffusa polysaccharide with plant exosome-loaded copper peptide, palmitoyl pentapeptide-4, and FGF2 growth peptide to form a complex delivery system. Through optimized preparation processes, a spray is formulated, achieving multi-mechanism synergy, highly efficient transdermal delivery, and long-lasting repair, providing a safer and more comprehensive new local treatment solution.

[0007] This application provides an active factor spray for inflammatory and allergic pruritus of the skin, wherein the spray is made from the following raw materials: 10-30 parts by weight of hyaluronic acid, 10-20 parts by weight of lycorine N-demethylated derivative, 5-15 parts by weight of Hedyotis diffusa polysaccharide, 30-50 parts by weight of purified water, and 0.1-5 parts by weight of exosome mixed peptides.

[0008] Preferably, the exosome mixed peptide is a mixture of mixed peptides and exosomes, wherein the mixed peptide is copper peptide, palmitoyl pentapeptide-4 and FGF2 growth peptide.

[0009] Preferably, the exosomes are plant exosomes.

[0010] Preferably, the plant exosomes are Acanthopanax senticosus exosomes.

[0011] Preferably, the preparation method of the lycorine N-demethylated derivative is as follows: lycorine and boron tribromide are reacted at a molar ratio of 1:3-1:4 in dichloromethane solvent at a volume of 8-10 times the mass of lycorine for 8-12 hours under nitrogen protection and magnetic stirring speed of 250-350 rpm at -10-0℃; after the reaction is completed, ice water is slowly added to quench the reaction, the pH is adjusted to 8-9 with sodium hydroxide solution, the organic phase is separated, dried with anhydrous sodium sulfate, and purified by column chromatography with dichloromethane:methanol = 10:1 as the eluent to obtain the lycorine N-demethylated derivative.

[0012] Preferably, the preparation method of the exosome mixed peptide is as follows: copper peptide, palmitoyl pentapeptide-4 and FGF2 growth peptide are dissolved in PBS at a mass ratio of 1:1:1, mixed, and then a suspension of Acanthopanax senticosus exosomes is added. The mixture is then subjected to low-voltage electroporation combined with short-time acoustic wave treatment to obtain the exosome mixed peptide.

[0013] Preferably, the preparation method of the Acanthopanax senticosus exosomes is as follows: select fresh Acanthopanax senticosus root bark, remove impurities, cut into pieces, soak in PBS, incubate at low temperature, filter, and centrifuge to obtain Acanthopanax senticosus exosomes.

[0014] Preferably, the low-temperature incubation is specifically a low-temperature incubation at 4°C for 12 hours.

[0015] Preferably, the preparation method of the active factor spray is as follows: (1) Preparation of aqueous base solution: Add purified water to a sterile reactor, stir at low speed, slowly add sieved hyaluronic acid while stirring to prevent clumping, and continue stirring until the hyaluronic acid is completely dissolved to obtain hyaluronic acid aqueous solution.

[0016] (2) Dissolving Hedyotis diffusa polysaccharide: While maintaining the stirring rate of the reactor, slowly add the sieved Hedyotis diffusa polysaccharide to the hyaluronic acid aqueous phase of step (1), and continue stirring to obtain the Hedyotis diffusa hyaluronic acid aqueous phase.

[0017] (3) Compounding: Add lycorine N-demethylated derivative and stir until there are no obvious particles; add exosome mixed peptides and continue stirring to fully integrate the peptide components with the system to obtain the compound stock solution.

[0018] (4) Homogenization and filtration: The composite stock solution is transferred to a high-pressure homogenizer for homogenization, sterilized by microporous membrane filtration, and the clarified stock solution after filtration is collected to obtain the spray semi-finished product.

[0019] (5) Filling and sealing: In a sterile filling environment, the finished liquid is filled into the spray bottle, the atomizing nozzle is immediately tightened and sealed to obtain the spray product.

[0020] Preferably, the pore size of the microporous filter membrane in step (4) is 0.45 μm or 0.22 μm.

[0021] The beneficial effects of the embodiments in this application are as follows: A multi-mechanism synergistic approach achieves integrated and highly effective treatment of itching, inflammation, and repair: By scientifically combining lycorine N-demethylated derivatives with specific anti-inflammatory and neuromodulatory activities, Hedyotis diffusa polysaccharide with immunomodulatory effects, and highly moisturizing hyaluronic acid with multiple functional peptides (blue copper peptide, palmitoyl pentapeptide-4, and FGF2 growth peptide) loaded on exosomes, this approach achieves synergistic effects on multiple targets, including inhibiting the release of inflammatory mediators, regulating neurosensitization signals, repairing the skin's physical barrier, and promoting cell regeneration, effectively breaking the vicious cycle of itching and scratching. Animal experiments have shown that this spray has an itching inhibition rate of 74.4%-78.6%, significant anti-inflammatory effects, and reduces serum IL-4 and TNF-α levels.

[0022] High safety, avoiding the adverse reactions of traditional hormone drugs: The core active ingredient of this application, lycorine N-demethylated derivative, is a non-hormonal anti-inflammatory substance that does not act on glucocorticoid receptors, fundamentally avoiding the side effects such as skin atrophy and telangiectasia that may be caused by long-term use of hormones. No adverse reactions such as skin redness and atrophy were observed in any of the example groups during the test, demonstrating higher safety.

[0023] The complete compound system has a clear synergistic effect. The absence of Hedyotis diffusa polysaccharide will weaken the immune regulation and auxiliary repair effects; the absence of the entire exosome mixed peptide system will result in the antipruritic effect and barrier repair ability being reduced to the minimum; the absence of FGF2 growth peptide will affect the rapid repair and regeneration of the skin. This proves that the present application has achieved synergistic effect through the scientific combination of each component. Detailed Implementation

[0024] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0025] In the following examples, the polysaccharide of Hedyotis diffusa (HPLC ≥ 90%) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; the FGF2 growth peptide sequence was Nap-FFGGPANVET, synthesized by Sangon Biotech (Shanghai) Co., Ltd.; copper peptide and palmitoyl pentapeptide-4 were purchased from Shandong Jitai Biotechnology Co., Ltd.; the IL-4 ELISA kit and (TNF-α) ELISA kit were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.; and all other reagents, unless otherwise specified, were obtained commercially available.

[0026] Preparation Example 1 Preparation of exosomes from Acanthopanax senticosus Preparation of exosome-like nanovesicles derived from Acanthopanax senticosus root bark: Fresh Acanthopanax senticosus root bark was selected, and the surface mud and impurities were thoroughly washed with double-distilled water. The bark was then cut into pieces approximately 0.5 cm in size. 2 Small pieces were soaked in PBS at a mass-to-volume ratio of 1:15 (g / mL) and incubated at 4°C for 12 h. After coarse filtration, the mixture was centrifuged at 1500×g for 30 min, filtered through 70 μm and 40 μm filters to remove large particulate impurities. The supernatant was further centrifuged at 4000×g for 30 min, filtered through a 0.7 μm filter, and centrifuged at 10000×g for 60 min to remove non-vesicular macromolecular impurities. The supernatant was then filtered through a 0.45 μm filter and ultracentrifuged at 100000×g for 70 min to precipitate exosome-like nanovesicles. The initial extract was centrifuged at 20%, 30%, 45%, and 60% sucrose density gradients at 100,000 × g, 4°C, and 150 min. The exosome-like nanovesicle fraction with 30%–45% sucrose layer was collected, diluted with PBS, and centrifuged again at 100,000 × g for 70 min to remove sucrose. After resuspending the precipitate, purified exosome-like nanovesicles derived from Acanthopanax senticosus were obtained and stored at 4°C for later use.

[0027] Identification of exosomes from Acanthopanax senticosus: 10 μL of exosome nanovesicle suspension derived from Acanthopanax senticosus was mixed with an equal volume of PBS and dropped onto a carbon membrane copper grid. The mixture was allowed to stand at room temperature for 3 min, stained with 2% uranium dioxide for 5 min, gently rinsed with double-distilled water, and dried at room temperature for 15 min. Vesicle morphology was then observed using a transmission electron microscope at 80-120 kV. Particle size analysis was performed using a nanoparticle tracking analyzer. Each sample was analyzed three times, and the average particle size and particle size distribution were statistically analyzed.

[0028] Transmission electron microscopy revealed that the successfully extracted exosome-like nanovesicles derived from Acanthopanax senticosus exhibited typical cup-shaped or disc-shaped structures. The vesicles were intact, clearly defined, and relatively uniform in size, consistent with the morphological characteristics of plant-derived exosome-like nanovesicles. Nanoparticle tracking analysis determined the concentration of Acanthopanax senticosus-derived exosome-like nanovesicles to be approximately 4.09 × 10⁻⁶. 10 The particle size distribution was 98.8-198 nm, with an average particle size of 116 nm. The particle size distribution curve was basically normally distributed, indicating that the sample had high homogeneity. The results showed that exosomes with typical morphology and suitable particle size were successfully isolated and obtained.

[0029] Preparation Example 2 Preparation of N-demethylated derivatives of lycorine Take 10g of lycorine (purity ≥99.5%) and add 80mL of dichloromethane to a 250mL dry round-bottom flask. Stir until completely dissolved. Place the flask in a -10℃ low-temperature cooling bath, connect a condenser, and purge the air in the system with nitrogen gas, maintaining a continuous and slow flow of nitrogen (50mL / min). Slowly add 43.2g of boron tribromide (purity ≥99.8%) through a syringe, ensuring the reaction system temperature does not exceed 0℃. Turn on the magnetic stirrer and adjust the stirring speed to 250rpm. The reaction continues for 8 hours. During the reaction, the progress is monitored every 2 hours using thin-layer chromatography (TLC) with dichloromethane:methanol = 10:1 as the developing solvent. Observe the disappearance of the lycorine starting material spots on the plate. At the same time, high-performance liquid chromatography (HPLC) can be used to assist in monitoring the reaction process to ensure complete reaction.

[0030] After the reaction is complete, the reaction solution is slowly poured into a beaker containing 200 mL of ice water to quench the reaction. During this process, the mixture is stirred continuously to avoid local overheating. The pH is adjusted to 8-9. The neutralized mixture is then transferred to a separatory funnel, allowed to stand and separate into layers, and the lower organic phase is separated. The organic phase is then transferred to an Erlenmeyer flask, and about 10 g of anhydrous sodium sulfate is added. The mixture is shaken for 10 min to dry. After the solution is allowed to stand until it is clear, the sodium sulfate solid is removed by filtration.

[0031] Preparation of silica gel column: Take 300g of 100-200 mesh silica gel and pack it into the column using the wet packing method with dichloromethane, ensuring that there are no air bubbles in the column, and test the column efficiency.

[0032] Sample loading and elution: Concentrate the dried organic phase to about 10 mL and carefully add it to the top of the silica gel column. Elute with a dichloromethane:methanol = 10:1 eluent at a flow rate of 1-2 drops / second. During elution, the composition of the eluent should be monitored in real time to avoid loss of the target product.

[0033] Collection and concentration: The eluent containing the target product was collected by HPLC, and the solvent was removed by vacuum distillation after combining the eluents to obtain 6g of lycorine N-demethylated derivative solid with a purity of 97%.

[0034] Preparation Example 3 Preparation of exosomal mixed peptides Preparation of single peptide solution: Weigh copper peptide, palmitoyl pentapeptide-4, and FGF2 growth peptide in a mass ratio of 1:1:1, add them separately to sterile 0.01mol / L PBS (pH 7.2~7.4), and dissolve them by magnetic stirring (100~200rpm, 4℃) to prepare a single peptide stock solution with a concentration of 1~5mg / mL; among them, palmitoyl pentapeptide-4 is slightly more lipid-soluble, and the stirring and dissolution time can be extended to 10~15min to ensure that there are no visible particles and avoid undissolved peptide fragments affecting the loading effect.

[0035] Preparation of mixed peptide solution: The above-prepared blue copper peptide, palmitoyl pentapeptide-4, and FGF2 growth peptide stock solutions were transferred to a sterile 50mL centrifuge tube at a volume ratio of 1:1:1. The mixture was gently pipetted 10-15 times (avoiding foaming) to obtain a homogeneous mixed peptide solution. The solution was temporarily stored at 4℃ in the dark and the subsequent loading operation was completed within 30 minutes.

[0036] Incubation of exosomes and mixed peptides: The exosome suspension of Acanthopanax senticosus was mixed with the above mixed peptide solution at a volume ratio of 5:1, with exosomes as the main component and peptides as the active ingredients to be loaded. The centrifuge tube was gently inverted 10 times to mix, and then placed in a constant temperature incubator at 4℃ and incubated in the dark for 30 min to allow the peptides to initially bind to the surface of the exosomes, which is a pretreatment for subsequent electroporation loading.

[0037] Low-voltage electroporation: Slowly transfer the incubated exosome-mixed peptide mixture into a pre-cooled 0.2cm gap sterile electroporation cup, avoiding the formation of air bubbles, as these can cause uneven current distribution during electroporation and damage the exosomes. Place the electroporation cup in an ice bath for 5 minutes, connect it to the electroporator, and set the low-voltage electroporation parameters: voltage: 100V, capacitance: 25μF, resistance: ∞ (infinite), number of pulses: 1~2, pulse duration: 8ms / pulse. Start the electroporator to complete the treatment. After treatment, continue to place the electroporation cup in an ice bath for 10 minutes to allow the micropores of the exosome membrane to close, ensuring that the loaded peptides are encapsulated within the exosomes or bound to the membrane surface, thus improving the stability of the load.

[0038] Short-term acoustic treatment: The electroporated exosome-peptide mixture was transferred to a sterile centrifuge tube in an ice bath and subjected to short-term acoustic treatment using an ultrasonic cell disruptor. The parameters were set as follows: power: 120W, working mode: intermittent: 2s on, 3s off, total treatment time: 1~2min; ice bath throughout to avoid the system temperature rising due to acoustic heat generation, ≤10℃. Short-term low-power acoustic treatment can evenly disperse unbound peptides and exosomes, further improving local loading efficiency, while avoiding exosome aggregation.

[0039] Post-processing and storage: Transfer the ultrasonically treated mixture to a sterile centrifuge tube and centrifuge at 3000 rpm for 5 minutes at 4°C to remove any exosome aggregates or impurities generated during the operation. Take the supernatant, which is the finished exosome mixed peptide product. Store at 4°C in a sealed container away from light to avoid repeated freeze-thaw cycles.

[0040] Preparation Example 4 Preparation of exosome mixed peptides (FGF2-free growth peptides) Preparation of single peptide solution: Weigh copper peptide and palmitoyl pentapeptide-4 at a mass ratio of 1:1, add sterile 0.01mol / L PBS (pH 7.2~7.4) to dissolve, and stir magnetically (100~200rpm, 4℃) to prepare a single peptide stock solution with a concentration of 1~5mg / mL; among them, palmitoyl pentapeptide-4 is slightly more lipid-soluble, and the stirring and dissolution time can be extended to 10~15min to ensure that there are no visible particles and avoid undissolved peptide fragments affecting the loading effect.

[0041] Preparation of mixed peptide solution: Transfer the above-prepared blue copper peptide and palmitoyl pentapeptide-4 stock solutions to a sterile 50mL centrifuge tube at a 1:1 volume ratio. Gently pipette 10-15 times (avoiding foaming) to obtain a homogeneous mixed peptide solution. Store at 4℃ in the dark for 30 minutes and complete the subsequent loading operation.

[0042] Incubation of exosomes and mixed peptides: The exosome suspension of Acanthopanax senticosus was mixed with the above mixed peptide solution at a volume ratio of 5:1, with exosomes as the main component and peptides as the active ingredients to be loaded. The centrifuge tube was gently inverted 10 times to mix, and then placed in a constant temperature incubator at 4℃ and incubated in the dark for 30 min to allow the peptides to initially bind to the surface of the exosomes, which is a pretreatment for subsequent electroporation loading.

[0043] Low-voltage electroporation treatment: Same as preparation example 3.

[0044] Short-time acoustic wave processing: Same as preparation example 3.

[0045] Post-processing and storage: Same as preparation example 3.

[0046] Example 1 Active ingredient spray for inflammatory and allergic itching of the skin Raw materials: 10 parts by weight of hyaluronic acid, 10 parts by weight of lycorine N-demethylated derivative, 5 parts by weight of Hedyotis diffusa polysaccharide, 30 parts by weight of purified water, and 0.1 parts by weight of exosome mixed peptide (preparation example 3).

[0047] Preparation method: Preparation of aqueous base solution: Add the above weight of purified water to a sterile reactor, stir at a low speed of 400 r / min, slowly add the sieved hyaluronic acid while stirring to prevent clumping, and continue stirring for 40 min after the addition is complete until the hyaluronic acid is completely dissolved to obtain the hyaluronic acid aqueous solution.

[0048] Dissolving Hedyotis diffusa polysaccharide: Keep the stirring rate of the reactor at 400 r / min, slowly add the sieved Hedyotis diffusa polysaccharide to the hyaluronic acid aqueous phase of step (1) above, continue stirring at 600 r / min for 30 min until the polysaccharide is completely dissolved, the solution remains clear and free of precipitate, and the temperature is controlled at ≤28℃ throughout the process to obtain the Hedyotis diffusa hyaluronic acid aqueous phase.

[0049] Compounding: Reduce the temperature of the reactor to 15-20℃, stir at a low speed of 300r / min, add the N-demethylated derivative of lycorine and stir until there are no obvious particles. Add it to the above mixture in 3-4 portions, stirring for 5 minutes after each addition until there are no obvious particles. After all the addition is completed, continue stirring for 15-20 minutes to ensure that the derivative is evenly dispersed and there is no local precipitation. Continue to maintain the temperature of the reactor at 15-20℃, reduce the stirring speed to 200r / min, and slowly add the exosome mixed peptides dropwise to the mixture. Continue stirring for 15 minutes to fully integrate the peptide components with the system to obtain the compound stock solution. The entire process should be carried out in the dark.

[0050] Homogenization and filtration: Transfer the above composite stock solution to a high-pressure homogenizer and homogenize it 2-3 times at 30MPa pressure. After each homogenization, let it stand for 5 minutes to fully break down the small particles in the system and obtain a uniform and transparent spray stock solution. After homogenization, the temperature should still be controlled ≤25℃. The homogenized stock solution is then filtered and sterilized by passing it through 0.45μm and 0.22μm microporous membranes in sequence. During filtration, the pressure should be kept stable to avoid foaming. Collect the clarified stock solution after filtration to obtain the spray semi-finished product.

[0051] Filling and sealing: In a sterile filling environment, the finished liquid is filled into the spray bottle, the atomizing nozzle is immediately tightened and sealed. During filling, avoid contact between the original liquid and the outside of the bottle opening to prevent contamination and obtain the finished spray product.

[0052] Example 2 Active ingredient spray for inflammatory and allergic itching of the skin Raw materials: 30 parts by weight of hyaluronic acid, 20 parts by weight of lycorine N-demethylated derivative, 15 parts by weight of Hedyotis diffusa polysaccharide, 50 parts by weight of purified water, and 5 parts by weight of exosome mixed peptides (preparation example 3).

[0053] Preparation method: Same as in Example 1.

[0054] Example 3 Active ingredient spray for inflammatory and allergic itching of the skin Raw materials: 20 parts by weight of hyaluronic acid, 15 parts by weight of lycorine N-demethylated derivative, 10 parts by weight of Hedyotis diffusa polysaccharide, 40 parts by weight of purified water, and 2.5 parts by weight of exosome mixed peptides (preparation example 3).

[0055] Preparation method: Same as in Example 1.

[0056] Comparative Example 1 Active ingredient spray for inflammatory and allergic itching of the skin Raw materials: 20 parts by weight of hyaluronic acid, 15 parts by weight of lycorine N-demethylated derivative, 40 parts by weight of purified water, and 2.5 parts by weight of exosome mixed peptide (preparation example 3).

[0057] Preparation method: Preparation of aqueous base solution: Add the above weight of purified water to a sterile reactor, stir at a low speed of 400 r / min, slowly add the sieved hyaluronic acid while stirring to prevent clumping, and continue stirring for 40 min after the addition is complete until the hyaluronic acid is completely dissolved to obtain the hyaluronic acid aqueous solution.

[0058] Compounding: Reduce the temperature of the reactor to 15-20℃, stir at a low speed of 300r / min, add the N-demethylated derivative of lycorine and stir until there are no obvious particles. Add it to the above mixture in 3-4 portions, stirring for 5 minutes after each addition until there are no obvious particles. After all the addition is completed, continue stirring for 15-20 minutes to ensure that the derivative is evenly dispersed and there is no local precipitation. Continue to maintain the temperature of the reactor at 15-20℃, reduce the stirring speed to 200r / min, and slowly add the exosome mixed peptides dropwise to the mixture. Continue stirring for 15 minutes to fully integrate the peptide components with the system to obtain the compound stock solution. The entire process should be carried out in the dark.

[0059] Homogenization and filtration: Transfer the above composite stock solution to a high-pressure homogenizer and homogenize it 2-3 times at 30MPa pressure. After each homogenization, let it stand for 5 minutes to fully break down the small particles in the system and obtain a uniform and transparent spray stock solution. After homogenization, the temperature should still be controlled ≤25℃. The homogenized stock solution is then filtered and sterilized by passing it through 0.45μm and 0.22μm microporous membranes in sequence. During filtration, the pressure should be kept stable to avoid foaming. Collect the clarified stock solution after filtration to obtain the spray semi-finished product.

[0060] Filling and sealing: In a sterile filling environment, the finished liquid is filled into the spray bottle, the atomizing nozzle is immediately tightened and sealed. During filling, avoid contact between the original liquid and the outside of the bottle opening to prevent contamination and obtain the finished spray product.

[0061] Comparative Example 2 Active ingredient spray for inflammatory and allergic itching of the skin Ingredients: 20 parts by weight of hyaluronic acid, 15 parts by weight of lycorine N-demethylated derivative, 10 parts by weight of Hedyotis diffusa polysaccharide, and 40 parts by weight of purified water.

[0062] Preparation method: Preparation of aqueous base solution: Add the above weight of purified water to a sterile reactor, stir at a low speed of 400 r / min, slowly add the sieved hyaluronic acid while stirring to prevent clumping, and continue stirring for 40 min after the addition is complete until the hyaluronic acid is completely dissolved to obtain the hyaluronic acid aqueous solution.

[0063] Dissolving Hedyotis diffusa polysaccharide: Keep the stirring rate of the reactor at 400 r / min, slowly add the sieved Hedyotis diffusa polysaccharide to the hyaluronic acid aqueous phase of step (1) above, continue stirring at 600 r / min for 30 min until the polysaccharide is completely dissolved, the solution remains clear and free of precipitate, and the temperature is controlled at ≤28℃ throughout the process to obtain the Hedyotis diffusa hyaluronic acid aqueous phase.

[0064] Compound preparation: Reduce the temperature of the reaction vessel to 15-20℃, stir at a low speed of 300r / min, add the N-demethylated derivative of lycorine and stir until there are no obvious particles. Add it to the above mixture in 3-4 portions, stirring for 5 minutes after each addition until there are no obvious particles. After all the addition is completed, continue stirring for 15-20 minutes to ensure that the derivative is evenly dispersed and there is no local precipitation; thus obtaining the composite stock solution.

[0065] Homogenization and filtration: Transfer the above composite stock solution to a high-pressure homogenizer and homogenize it 2-3 times at 30MPa pressure. After each homogenization, let it stand for 5 minutes to fully break down the small particles in the system and obtain a uniform and transparent spray stock solution. After homogenization, the temperature should still be controlled ≤25℃. The homogenized stock solution is then filtered and sterilized by passing it through 0.45μm and 0.22μm microporous membranes in sequence. During filtration, the pressure should be kept stable to avoid foaming. Collect the clarified stock solution after filtration to obtain the spray semi-finished product.

[0066] Filling and sealing: In a sterile filling environment, the finished liquid is filled into the spray bottle, the atomizing nozzle is immediately tightened and sealed. During filling, avoid contact between the original liquid and the outside of the bottle opening to prevent contamination and obtain the finished spray product.

[0067] Comparative Example 3 Active ingredient spray for inflammatory and allergic itching of the skin Raw materials: 20 parts by weight of hyaluronic acid, 15 parts by weight of lycorine N-demethylated derivative, 10 parts by weight of Hedyotis diffusa polysaccharide, 40 parts by weight of purified water, and 2.5 parts by weight of exosome mixed peptides (preparation example 4).

[0068] Preparation method: Preparation of aqueous base solution: Add the above weight of purified water to a sterile reactor, stir at a low speed of 400 r / min, slowly add the sieved hyaluronic acid while stirring to prevent clumping, and continue stirring for 40 min after the addition is complete until the hyaluronic acid is completely dissolved to obtain the hyaluronic acid aqueous solution.

[0069] Dissolving Hedyotis diffusa polysaccharide: Keep the stirring rate of the reactor at 400 r / min, slowly add the sieved Hedyotis diffusa polysaccharide to the hyaluronic acid aqueous phase of step (1) above, continue stirring at 600 r / min for 30 min until the polysaccharide is completely dissolved, the solution remains clear and free of precipitate, and the temperature is controlled at ≤28℃ throughout the process to obtain the Hedyotis diffusa hyaluronic acid aqueous phase.

[0070] Compounding: Reduce the temperature of the reactor to 15-20℃, stir at a low speed of 300r / min, add the N-demethylated derivative of lycorine and stir until there are no obvious particles. Add it to the above mixture in 3-4 portions, stirring for 5 minutes after each addition until there are no obvious particles. After all the addition is completed, continue stirring for 15-20 minutes to ensure that the derivative is evenly dispersed and there is no local precipitation. Continue to maintain the temperature of the reactor at 15-20℃, reduce the stirring speed to 200r / min, and slowly add the exosome mixed peptides dropwise to the mixture. Continue stirring for 15 minutes to fully integrate the peptide components with the system to obtain the compound stock solution. The entire process should be carried out in the dark.

[0071] Homogenization and filtration: Transfer the above composite stock solution to a high-pressure homogenizer and homogenize it 2-3 times at 30MPa pressure. After each homogenization, let it stand for 5 minutes to fully break down the small particles in the system and obtain a uniform and transparent spray stock solution. After homogenization, the temperature should still be controlled ≤25℃. The homogenized stock solution is then filtered and sterilized by passing it through 0.45μm and 0.22μm microporous membranes in sequence. During filtration, the pressure should be kept stable to avoid foaming. Collect the clarified stock solution after filtration to obtain the spray semi-finished product.

[0072] Filling and sealing: In a sterile filling environment, the finished liquid is filled into the spray bottle, the atomizing nozzle is immediately tightened and sealed. During filling, avoid contact between the original liquid and the outside of the bottle opening to prevent contamination and obtain the finished spray product.

[0073] Test case Active Factor Spray Anti-inflammatory, Antipruritic, and Skin Repair Trial Experimental animals: 80 SPF-grade BALB / c allergic contact dermatitis pruritus model mice and 10 SPF-grade BALB / c blank nude mice, half male and half female, weighing 20-25g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were allowed free access to food, and the room temperature was maintained at 20-26℃ and the humidity at 45%-70%. They were used for experiments after 1 week of acclimatization feeding.

[0074] Experimental grouping and drug administration (10 animals per group): Example 1 Group: Spray the back and right ear with the spray from Example 1, one press of the spray (0.1 mL), twice a day for 7 consecutive days; Example 2 group: Spray the back and right ear with the spray from Example 2, one press of the spray (0.1 mL), twice a day for 7 consecutive days; Example 3 group: Spray the back and right ear with the spray from Example 3, one press of the spray (0.1 mL), twice a day for 7 consecutive days; Comparative Example 1: Spray Comparative Example 1 spray onto the back and right ear, using a single press of the spray pump (0.1 mL), twice daily for 7 consecutive days; Comparative Example 2: Spray the back and right ear with the Comparative Example 2 spray, one press of the spray (0.1 mL), twice a day for 7 consecutive days; Comparative Example 3: Spray the back and right ear with Comparative Example 3 spray, one press of the spray (0.1 mL), twice a day for 7 consecutive days; Model group: No treatment; Positive control group: 0.1% hydrocortisone cream; Blank control group: Apply physiological saline.

[0075] Test protocol: Evaluation of antipruritic effect: On days 1, 3 and 7 after administration, the number of times mice scratched within 1 hour was recorded using an electronic scratch counter, and the scratch inhibition rate was calculated (inhibition rate = (number of scratches in the model group - number of scratches in the administration group) / number of scratches in the model group × 100%); the blank control group only recorded the number of scratches during normal activities (no pathological itching).

[0076] Anti-inflammatory activity assay: On day 7 of drug administration, blood was collected by enucleation, serum was separated by centrifugation, and the levels of IL-4 and TNF-α in the serum were detected by ELISA kit; the swelling degree of the right ear of mice was measured (ear thickness was measured by vernier calipers, swelling degree = ear thickness after stimulation - ear thickness before stimulation); the serum inflammatory factor levels and the basic ear thickness were detected simultaneously in the blank control group.

[0077] Skin barrier repair test: On day 7 of drug administration, the transepidermal water loss (TEWL) of the hairless area on the back of mice was measured using a skin barrier function tester. The lower the TEWL value, the better the skin barrier repair. The baseline TEWL value of the back skin was measured in the blank control group.

[0078] Safety observation: Observe the mice in each group daily for adverse reactions such as redness, swelling, peeling, and erosion of the skin, and record the incidence rate.

[0079] Experimental data: Statistical analysis was performed using GraphPad Prism 9.0.1, and the results were validated using the Shapiro-Wilk test. The results showed that all variables conformed to a normal distribution. Therefore, the research data were used as the basis for the study. Presented in ±s form. For multiple groups of data that conform to a normal distribution and have homogeneity of variance, one-way ANOVA was used. If the difference was statistically significant (P < 0.05), Bonferroni multiple tests were used for pairwise comparisons between groups. If the data did not meet the homogeneity of variance requirement, Tamhane's T² test was used instead. Independent samples t-tests (Student's t-test) were used to compare two groups of data, with P < 0.05 indicating a statistically significant difference. Data are shown in Table 1. Table 1 Experimental Data Experimental results: Groups 1-3 showed significant improvements in itching inhibition rate (74.4%-78.6%), ear swelling inhibition, and skin barrier repair (TEWL value 14.2-18.3 g / h·m). 2 All three formulations showed excellent and balanced effects, significantly superior to each comparative group, and comparable to the positive control group (0.1% hydrocortisone cream); *: vs model group P<0.05. Comparative Example 1 (lacking Hedyotis diffusa polysaccharide): showed weaker performance in anti-inflammatory (higher IL-4, TNF-α) and barrier repair (higher TEWL), indicating that Hedyotis diffusa polysaccharide makes an important contribution to immune regulation and auxiliary repair. Comparative Example 2 (lacking exosome mixed peptides): showed the worst antipruritic effect (inhibition rate 52.3%) and the worst barrier repair ability (highest TEWL value) among all comparative examples, highlighting the key role of the exosome delivery system and the complex of copper peptide, palmitoyl pentapeptide-4, and FGF2 growth peptide in achieving deep repair and enhancing the durability of antipruritic effects; Comparative Example 3 (lacking FGF2 growth peptide): its repair effect (TEWL value) was second only to the complete example, indicating that FGF2 growth peptide has irreplaceable value in promoting rapid skin repair and regeneration.

[0080] In summary, this application has successfully developed an active factor spray for inflammatory and allergic pruritus of the skin. Through the innovative combination of multi-component scientific compounding and exosome targeted delivery system, it achieves integrated synergistic effects of relieving itching, anti-inflammation, and repair. In animal models, this spray has shown antipruritic and anti-inflammatory efficacy comparable to 0.1% hydrocortisone cream, while completely avoiding adverse reactions such as skin atrophy caused by hormone drugs. Its safety is significantly better than traditional hormone therapy.

[0081] The experimental results show that the components in this formula have clear functions and synergistic effects: the N-demethylated derivative of lycorine exerts non-hormonal anti-inflammatory and neuromodulatory effects; Hedyotis diffusa polysaccharide enhances immune regulation; exosome-loaded copper peptide, palmitoyl pentapeptide-4, and FGF2 growth peptide play key roles in repairing the skin barrier and promoting cell regeneration; and Acanthopanax senticosus exosomes, as highly efficient delivery carriers, significantly improve the transdermal absorption and targeting of active ingredients. The absence of any key component will lead to a decrease in overall efficacy, confirming the rationality and necessity of this compound design.

[0082] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. An active ingredient spray for inflammatory and allergic pruritus of the skin, characterized in that, The spray is made from the following raw materials: 10-30 parts by weight of hyaluronic acid, 10-20 parts by weight of lycorine N-demethylated derivative, 5-15 parts by weight of Hedyotis diffusa polysaccharide, 30-50 parts by weight of purified water, and 0.1-5 parts by weight of exosome mixed peptides.

2. The active ingredient spray for inflammatory and allergic pruritus of the skin as described in claim 1, characterized in that, The exosome mixed peptide is a mixture of mixed peptides and exosomes, wherein the mixed peptide is copper peptide, palmitoyl pentapeptide-4 and FGF2 growth peptide.

3. The active ingredient spray for inflammatory and allergic pruritus of the skin as described in claim 2, characterized in that, The exosomes are plant exosomes.

4. The active ingredient spray for inflammatory and allergic pruritus of skin as described in claim 3, characterized in that, The plant exosomes were Acanthopanax senticosus exosomes.

5. The active ingredient spray for inflammatory and allergic pruritus of the skin as described in claim 1, characterized in that, The preparation method of the lycorine N-demethylated derivative is as follows: Lycorine and boron tribromide are reacted at a molar ratio of 1:3-1:4 in dichloromethane solvent at a volume of 8-10 times the mass of lycorine for 8-12 hours under nitrogen protection and magnetic stirring speed of 250-350 rpm at -10-0℃. After the reaction is completed, ice water is slowly added to quench the reaction, and the pH is adjusted to 8-9 with sodium hydroxide solution. The organic phase is separated, dried with anhydrous sodium sulfate, and purified by column chromatography with dichloromethane:methanol = 10:1 as the eluent to obtain the lycorine N-demethylated derivative.

6. The active ingredient spray for inflammatory and allergic pruritus of the skin as described in claim 1, characterized in that, The preparation method of the exosome mixed peptide is as follows: copper peptide, palmitoyl pentapeptide-4 and FGF2 growth peptide are dissolved in PBS at a mass ratio of 1:1:1, mixed, and then eleutherococcus senticosus exosome suspension is added. The mixture is then subjected to low-voltage electroporation combined with short-time acoustic wave treatment to obtain the exosome mixed peptide.

7. The active ingredient spray for inflammatory and allergic pruritus of skin as described in claim 4, characterized in that, The preparation method of the Acanthopanax bark exosomes is as follows: select fresh Acanthopanax bark root bark, remove impurities, cut into pieces, soak in PBS, incubate at low temperature, filter, and centrifuge to obtain Acanthopanax bark exosomes.

8. The active ingredient spray for inflammatory and allergic pruritus of skin as described in claim 7, characterized in that, The low-temperature incubation specifically refers to incubation at 4°C for 12 hours.

9. The active ingredient spray for inflammatory and allergic pruritus of the skin as described in claim 1, characterized in that, The preparation method of the active factor spray is as follows: Preparation of aqueous base solution: Add purified water to a sterile reaction vessel, stir at low speed, slowly add sieved hyaluronic acid while stirring to prevent clumping, and continue stirring after adding until the hyaluronic acid is completely dissolved to obtain hyaluronic acid aqueous solution. Dissolving Hedyotis diffusa polysaccharide: While maintaining the stirring rate of the reactor, slowly add the sieved Hedyotis diffusa polysaccharide to the hyaluronic acid aqueous phase of step (1), and continue stirring to obtain the Hedyotis diffusa hyaluronic acid aqueous phase. Compound formulation: Add lycorine N-demethylated derivative and stir until no obvious particles are visible; add exosome mixed peptides and continue stirring to fully integrate the peptide components with the system to obtain the compound stock solution; Homogenization and filtration: The composite stock solution is transferred to a high-pressure homogenizer for homogenization, and then filtered through a microporous membrane for sterilization. The clarified stock solution after filtration is collected to obtain the spray semi-finished product. Filling and sealing: In a sterile filling environment, the finished liquid is filled into the spray bottle, the atomizing nozzle is immediately tightened and sealed to obtain the finished spray product.

10. The active ingredient spray for inflammatory and allergic pruritus of skin as described in claim 9, characterized in that, The microporous filter membrane in step (4) has a pore size of 0.45 μm and 0.22 μm.

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