Preparation method and application of self-assembled nano-preparation loaded with xanthoxylyl amide

Abstract

This invention belongs to the field of nanoparticle technology, proposing a method for preparing a self-assembled nanoparticle loaded with zanthoxylum bungeanum and its application. The method involves first extracting and purifying zanthoxylum bungeanum from the pericarp of Zanthoxylum bungeanum, then mixing the zanthoxylum bungeanum with glycyrrhizic acid at a certain mass ratio. Using ultrasonic dispersion, the hydrophobic interaction between the two substances physically encapsulates the zanthoxylum bungeanum within the glycyrrhizic acid-based self-assembled nanoparticle. This invention requires no chemical modification of the zanthoxylum bungeanum, and the preparation process is simple, green, and safe. The prepared nanoparticles have uniform particle size, good dispersibility, high encapsulation efficiency and drug loading rate, and significantly improve the thermal stability, light stability, and storage stability of zanthoxylum bungeanum, overcoming its easy degradation. This nanoparticle provides a foundation for the application of zanthoxylum bungeanum in the pharmaceutical, food, and cosmetic fields.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to the extraction of cinnamic acid and the self-assembled nano-formulation containing cinnamic acid. Background Technology

[0002] Sichuan pepper (Zanthoxylum bungeanum) is a plant belonging to the genus Zanthoxylum in the Rutaceae family and is a traditional Chinese medicinal herb used in both food and medicine. Zanthoxylum bungeanum amides are unique amide-type active components found in Sichuan pepper, mainly including hydroxy-α-salicornin, hydroxy-β-salicornin, and hydroxy-ε-salicornin. Studies have shown that zanthoxylum bungeanum amides possess various biological activities such as anti-inflammatory, antioxidant, anesthetic and analgesic, antibacterial, and hypoglycemic effects, and have broad application prospects in the pharmaceutical, food, and cosmetic fields. However, methods for large-scale extraction of zanthoxylum bungeanum amides from Sichuan pepper are still very limited. Traditional extraction methods yield products with many impurities and low purity, which restricts further research and application. Patent CN 119504476 A proposes a method for extracting amide components from Sichuan pepper using a natural eutectic solvent assisted by ultrafine grinding. After drying, Sichuan pepper is subjected to high-speed grinding and low-temperature ultrafine grinding to obtain ultrafine powder. Then, a natural eutectic solvent prepared with hydrogen bond acceptors and hydrogen bond donors is added for microwave-assisted extraction, which is repeated several times. The extract is then concentrated and filtered through a membrane. Extraction with eutectic solvents requires ultra-fine grinding of the Sichuan pepper raw material, which is a complex process. Furthermore, eutectic solvent extraction is still in the laboratory stage and cannot be scaled up for industrial production.

[0003] Although sanshinone has high bioactivity, its molecular structure contains conjugated triene bonds, making it highly sensitive to light, heat, and oxygen, resulting in extremely poor stability and severely limiting its practical applications. Public patent CN 115212169 A proposes a method for isolating sanshinone from Sichuan pepper and preparing nanoparticles: Sichuan pepper pericarp is dried and ground, and the crude amide product is extracted ultrasonically with dimethyl sulfoxide aqueous solution, followed by column chromatography purification to obtain sanshinone; using DMAP and DIPC as catalysts, esterification is used to couple sanshinone to carboxylic acid groups, which then self-assemble into nanoparticles in deionized water. This method requires column chromatography purification and organic catalytic reactions, is cumbersome, and relies on multiple organic reagents, making it unsuitable for industrial production. Therefore, developing a chemically-free, green, safe nanoparticle delivery system that can significantly improve the stability of sanshinone has significant practical implications and application value. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the first objective of this invention is to propose a method for preparing a self-assembled nano-formulation loaded with zanthoxylate.

[0005] The second objective of this invention is to provide a self-assembled nanoformulation loaded with zafylan obtained by the aforementioned preparation method.

[0006] A third objective of this invention is to propose the application of the self-assembled nanoformulations loaded with zanthamide.

[0007] The technical solution for achieving the above-mentioned objective of this invention is as follows: A method for preparing a self-assembled nanoparticle formulation loaded with zanthoxylate, comprising the following steps: (1) Using Sichuan pepper fruit peel powder as raw material, Sichuan pepper oil resin was obtained by supercritical CO2 extraction. (2) Dissolve the pepper oil resin in an ethanol aqueous solution, add macroporous weak acid cation exchange resin for decolorization treatment, filter, evaporate under reduced pressure to dryness, and obtain the decolorized product. (3) The decolorized product was loaded onto a polystyrene weakly polar adsorption resin column, eluted with methanol, the eluent was collected, and evaporated under reduced pressure to obtain crude zanthamide. (4) Dissolve the crude zanthoxylamide in methanol, separate it by preparative high performance liquid chromatography, collect the main peak fraction, evaporate it under reduced pressure and freeze dry to obtain zanthoxylamide; (5) Glycyrrhizic acid was added to water to prepare an aqueous solution of glycyrrhizic acid. After dissolving citric acid in ethanol, the solution was added dropwise to the aqueous solution of glycyrrhizic acid. The solution was then dispersed by ultrasonication, the ethanol was removed by rotary evaporation under reduced pressure, and the solution was filtered to obtain a self-assembled nano-formulation loaded with citric acid.

[0008] This invention selects supercritical CO2 extraction for the extraction of cinnamamide, mainly based on the following considerations: Compared with organic solvent extraction, supercritical CO2 extraction does not use organic solvents throughout the entire process, eliminating the risk of solvent residue, making it green and safe, and meeting the stringent requirements for raw materials in the pharmaceutical, food, and cosmetic fields; the low extraction temperature (45-55℃) effectively protects the thermally unstable conjugated structure of cinnamamide, preventing its degradation or isomerization; at the same time, supercritical CO2 has good selectivity for cinnamamide, which can reduce the co-extraction of impurities such as pigments and oils, reduce the burden of subsequent purification, and provide high-quality raw materials for the subsequent efficient self-assembly with glycyrrhizic acid.

[0009] A preferred technical solution of the present invention is that the conditions for supercritical CO2 extraction in step (1) are: extraction temperature 45~55℃, extraction pressure 20~30MPa, separation vessel I temperature 40~50℃, pressure 8~10MPa, separation vessel II pressure 3~5MPa, and extraction time 60~100min.

[0010] In step (2), the volume concentration of the ethanol aqueous solution is 60-80%; the mass ratio of macroporous weak acid cation exchange resin to pepper oil resin is 1.5-2.5:1; the decolorization conditions are: temperature 25-35℃, oscillation speed 100-200r / min, and time 8-16h.

[0011] In step (3), the aspect ratio of the macroporous adsorption resin column can be (10~20):1, and the elution flow rate is 1~4 mL / min. The resin can be one that is already available in the art, such as AB-8 resin.

[0012] Further, the conditions for the preparative high performance liquid chromatography described in step (4) are: C18 column, particle size 10 μm, inner diameter 10 mm, column length 250 mm; mobile phase 40~50% methanol-water; flow rate 2~4 mL / min.

[0013] When using a C18 column, select a detection wavelength of 260~280nm.

[0014] In step (5), the concentration of the glycyrrhizic acid aqueous solution is 1~5 mg / mL.

[0015] Glycyrrhizic acid is a natural amphiphilic triterpenoid saponin that can spontaneously assemble into nano-formations in aqueous solution. It physically encapsulates citric acid amide through hydrophobic interactions. Glycyrrhizic acid itself has anti-inflammatory and antioxidant activities, is widely available, and has high biosafety.

[0016] In step (5), the mass ratio of glycyrrhizic acid to citric acid amide is 3~13:1; the volume fraction of ethanol is 4~12%; the power of ultrasonic dispersion is 180~260W, and the time is 20~40min.

[0017] The filtration in step (5) is through a 0.45 μm microporous membrane.

[0018] More preferably, in step (5), glycyrrhizic acid is added to water and heated to 40~60°C to dissolve, wherein the mass ratio of glycyrrhizic acid to citric acid amide is 5:1; The volume fraction of ethanol is 8%; the power of ultrasonic dispersion is 220W.

[0019] The self-assembled nanoformulation loaded with zanthoxylate obtained by the preparation method described in this invention.

[0020] The application of the self-assembled nanoformulation loaded with citriamide described in this invention in the preparation of anti-inflammatory, antioxidant, or cell migration-promoting drugs or cosmetics.

[0021] This invention is the first to discover and utilize xanthocyanin (containing three homologues: hydroxy-α-xanthocyanin, hydroxy-β-xanthocyanin, and hydroxy-ε-xanthocyanin) extracted from Sichuan pepper, which physically self-assembles into micelles through hydrophobic interactions. Compared with using commercially available pure xanthocyanin, the mixed-state xanthocyanin extracted directly from natural Sichuan pepper by this invention significantly reduces costs and is more suitable for large-scale production. Furthermore, the three natural homologues coexist within the micelles, each exerting its own active effect, resulting in a more comprehensive overall function. In addition, this invention requires no chemical modification of the xanthocyanin, making the preparation process green and safe, overcoming the application bottleneck of xanthocyanin's easy degradation, and providing a technical foundation for its low-cost, large-scale application in the pharmaceutical, food, and cosmetic fields.

[0022] The beneficial effects of this invention are as follows: (1) The preparation method of this self-assembled nano-formulation loaded with citriamide does not require any chemical modification of citriamide. The preparation process is simple, green and safe. The prepared nano-formulation has uniform particle size and good dispersibility, with high encapsulation rate and drug loading rate. It also significantly improves the thermal stability, light stability and storage stability of citriamide, overcoming the defect of easy degradation of citriamide.

[0023] (2) The method proposed in this invention can easily and quickly separate and purify zanthoxylum amide from the crop Sichuan pepper. The separated and purified zanthoxylum amide has high purity and mainly contains hydroxy-α-santhioline, hydroxy-β-santhioline and hydroxy-ε-santhioline. (3) This invention utilizes the natural amphiphilic self-assembly characteristics of plant-based products, without the need for any chemical modification of the pepper amide. Physical encapsulation can be achieved through hydrophobic interactions, and the preparation process is green and safe. (4) The prepared glycyrrhizic acid self-assembled nano-formulation has uniform particle size and good dispersibility, and has high encapsulation rate and high drug loading rate; (5) The prepared nano-formulation significantly improved the thermal stability, light stability and storage stability of zanthoxylum amide, overcoming the defect of zanthoxylum amide being easily degraded.

[0024] (6) The prepared nano-formulation has good biocompatibility and biosafety, which provides a basis for the application of pepper amide in the fields of medicine, food and cosmetics.

[0025] (7) The method of the present invention is easy to implement and more efficient and controllable. Attached Figure Description

[0026] Figure 1 This is a high-performance liquid chromatogram of the zanthamide and zanthamide standard obtained by extraction and purification in this invention; Figure 2 The standard curve for the citriamide standard; Figure 3Particle size distribution diagram of glycyrrhizic acid micelles and xanthanamide-glycyrrhizic acid nanoparticles; Figure 4 Transmission electron microscopy images of glycyrrhizic acid micelles and xanthanamide-glycyrrhizic acid nanoparticles; Figure 5 This is a Fourier transform infrared spectrum; Figure 6 The X-ray diffraction pattern of the pepper amide-glycyrrhizic acid nano-formulation prepared in this invention; Figure 7 This is a comparison diagram of the thermal stability of the present invention's zanthoxylate-glycyrrhizic acid nano-formulation and zanthoxylate. Figure 8 This is a comparison diagram of the UV light stability of the present invention's zanthoxyl-glycyrrhizic acid nano-formulation and zanthoxyl amide; Figure 9 This is a comparison chart of the storage stability of the present invention's zanthoxylate-glycyrrhizic acid nano-formulation and zanthoxylate. Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0028] The present invention discloses a method for isolating xanthanin from Sichuan pepper and preparing it into a glycyrrhizic acid self-assembled nano-formulation, the specific implementation process of which is as follows: I. Extraction of Sichuan pepper oleoresin from Sichuan pepper pericarp using supercritical CO2 extraction: The specific implementation process of this step is as follows: The dried Sichuan pepper pericarps are pulverized, passed through a 10-20 mesh sieve, and placed in a supercritical CO2 extraction vessel. The extraction temperature is set to 45-55℃, and the extraction pressure to 20-30 MPa; the temperature of separation vessel I is 40-50℃, and the pressure is 8-10 MPa; the pressure of separation vessel II is 3-5 MPa. The extraction time is 60-100 minutes, and the Sichuan pepper oil and resin are collected from the outlet of the separation vessel.

[0029] II. Decolorization treatment of Sichuan pepper oil resin using D152 type macroporous adsorption resin: One specific implementation process of this step is as follows: Dissolve the pepper oil resin in an ethanol aqueous solution with a volume concentration of 60-80%, add D152 type macroporous adsorption resin, and the mass ratio of resin to pepper oil resin is (1.5-2.5):1. Place in a constant temperature shaking incubator and treat for 8-16 hours at a temperature of 25-35℃ and a shaking speed of 100-200 r / min. Filter under normal pressure, collect the filtrate, and evaporate to dryness under reduced pressure at 35-45℃ to obtain the decolorized product.

[0030] III. The decolorized product was purified by column chromatography using AB-8 macroporous adsorption resin to obtain crude xanthanamide: One specific implementation procedure for this step is as follows: The AB-8 macroporous adsorption resin is rinsed with deionized water until clear, then soaked in twice the volume of anhydrous ethanol for 20-30 hours to swell, and then washed with deionized water until no ethanol odor remains. The pretreated AB-8 resin is wet-packed into a glass chromatography column with a height-to-diameter ratio of (10-20):1. The decolorized product is dissolved in methanol and loaded onto the column. Elution is performed with methanol at a flow rate of 1-4 mL / min, and the eluent is collected in fractions. The eluents are combined and evaporated to dryness under reduced pressure at 35-45℃ to obtain crude xanthanamide.

[0031] IV. The crude zanthamide was separated and purified by preparative high-performance liquid chromatography to obtain zanthamide: One specific implementation procedure for this step is as follows: Crude xanthanamide is dissolved in methanol. A C18 chromatographic column (particle size 10 μm, inner diameter 10 mm, column length 250 mm) is prepared, using a methanol-water mixture with a volume fraction of 40–50% as the mobile phase, a flow rate of 2–4 mL / min, and a detection wavelength of 260–280 nm. Separation is performed at room temperature. The main peak fraction is collected, and the solvent is removed by rotary evaporation under reduced pressure at 35–45 °C. The fraction is then freeze-dried for 40–60 h to obtain xanthanamide.

[0032] V. Utilizing the self-assembly properties of glycyrrhizic acid, xanthanil was physically encapsulated within glycyrrhizic acid nanoparticles to prepare glycyrrhizic acid self-assembled nanoparticles loaded with xanthanil: One specific implementation process of this step is as follows: Glycyrrhizic acid is dissolved in distilled water and heated and stirred at 40-60℃ until completely dissolved to obtain an aqueous solution of glycyrrhizic acid. Piperamide is dissolved in ethanol with a volume fraction of 4-12% to obtain a piperamide ethanol solution. While stirring, the piperamide ethanol solution is added dropwise to the aqueous solution of glycyrrhizic acid, with a mass ratio of glycyrrhizic acid to piperamide of (3-13):1, and the volume fraction of ethanol in the mixture is 4-12%. Stirring continues for 0.5-2 hours. After cooling to room temperature, ultrasonic dispersion is performed in an ice-water bath using an ultrasonic cell disruptor with an ultrasonic power of 180-260W for 20-40 minutes. Then, the ethanol is removed by rotary evaporation under reduced pressure at 35-45℃, and distilled water is added to bring the total mass concentration of the nano-formulation to 0.1-0.5 wt%. The mixture is then filtered through a 0.45 μm microporous membrane to obtain the glycyrrhizic acid self-assembled nano-formulation loaded with piperamide.

[0033] The glycyrrhizic acid self-assembled nanoformulation loaded with zanthamide prepared in this invention has good stability and biocompatibility, and can be used as a drug or cosmetic formulation for anti-inflammatory, antioxidant or cell migration promotion.

[0034] Several examples are provided below. In these examples, the pepper pericarp was *Zanthoxylum bungeanum* (also known as Jiuyeqing pepper) from Jiangjin, Chongqing, which was dried and then vacuum-sealed at -20°C; glycyrrhizic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity ≥95%; methanol and acetonitrile were chromatographically pure and purchased from Thermo Fisher Scientific (China) Co., Ltd.; D152 and AB-8 macroporous adsorption resins were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] Unless otherwise specified, all methods used in this specification are existing techniques in the field.

[0036] Example 1

[0037] This embodiment provides a method for supercritical CO2 extraction of oleoresin from Sichuan pepper, the specific steps of which are as follows: 1) Crush the dried pepper peel of Jiuyeqing, pass it through a 10-mesh sieve, weigh 2.5 kg and place it in a supercritical CO2 extraction vessel.

[0038] 2) Set the extraction temperature to 50.5℃ and the extraction pressure to 25.5MPa; the temperature of separation vessel I is 45℃ and the pressure is 9MPa; the pressure of separation vessel II is 4MPa.

[0039] 3) Extract for 80 minutes, collect the pepper oil resin from the outlet of the separator, with a yield of about 8.5%.

[0040] Example 2

[0041] This embodiment provides the operation of decolorizing D152 resin and purifying AB-8 resin, and the specific steps are as follows: 1) Weigh 1.00g of the pepper oil resin obtained by the method in Example 1 and dissolve it in 40mL of 70% ethanol. Take 6mL of this solution into a 100mL Erlenmeyer flask and add 2.00g of D152 macroporous adsorption resin.

[0042] 2) Place the conical flask in a constant temperature shaking incubator and shake it at 30℃ and 150r / min for 12h.

[0043] 3) Filter under normal pressure, collect the filtrate, and evaporate to dryness under reduced pressure at 40°C to obtain the decolorized product.

[0044] 4) Rinse the AB-8 macroporous resin with deionized water until clear, soak it in two volumes of anhydrous ethanol for 24 hours to swell, and then wash it with deionized water until there is no ethanol odor. Wet pack the pretreated AB-8 resin into a glass chromatography column (2.5cm×40cm), with a column height of 21.5cm (height-to-diameter ratio of approximately 15:1).

[0045] 5) Weigh 0.50 g of the decolorized product and dissolve it in 5 mL of methanol, then load the sample onto the solvent. Elute with methanol at a flow rate of 2.5 mL / min, collecting one tube for every 50 mL eluent. Collect the eluent and evaporate it to dryness under reduced pressure at 40 °C to obtain crude xanthanamide.

[0046] Example 3

[0047] This example illustrates the preparative high-performance liquid chromatography (HPLC) purification of zanthamide. The specific steps are as follows: 1) Take 10 mg of the crude xanthanamide obtained by the method in Example 2 and dissolve it in 5 mL of methanol.

[0048] 2) Chromatographic conditions: SEPAXGP-C18 preparative column (10μm, 10mm×250mm); mobile phase: 45% methanol-water; flow rate: 3.0mL / min; detection wavelength: 270nm; room temperature.

[0049] 3) Collect the main peak fraction with a retention time of 11-15 min, evaporate it at 40℃ by rotary evaporation, and freeze-dry it for 48 h to obtain high-purity zinebamide.

[0050] Quantitative analysis was performed using HPLC (Zorbax SB-C18 analytical column, 4.6 mm × 250 mm × 5 μm; mobile phase: water:acetonitrile = 55:45; flow rate: 0.8 mL / min; detection wavelength: 270 nm; column temperature: 40 ℃). Results are as follows: Figure 1 As shown, the obtained product has the same retention time as the cinnamic acid standard. A standard curve for the standard was established. Figure 2 Calculations show that the total content of xanthocyanin is 850.34±36.66 mg / g, including 702.73±70.92 mg / g of hydroxy-α-xanthocyanin, 131.23±18.72 mg / g of hydroxy-β-xanthocyanin, and 16.38±2.97 mg / g of hydroxy-ε-xanthocyanin.

[0051] Example 4

[0052] This embodiment provides a self-assembled glycyrrhizic acid nanoparticle formulation loaded with citriamide, comprising the following experiments: A suitable amount of citriamide was weighed and dissolved in ethanol to obtain a solution with a concentration of 1 mg / mL, which was then added dropwise to an aqueous glycyrrhizic acid solution with a concentration of 5 mg / mL. The solution was then ultrasonically dispersed, subjected to reduced pressure rotary evaporation to remove ethanol, and filtered (through a 0.45 μm microporous membrane) to obtain the self-assembled glycyrrhizic acid nanoparticle formulation loaded with citriamide. The aqueous glycyrrhizic acid solution was prepared by weighing a suitable amount of glycyrrhizic acid and stirring in distilled water at 50°C (500 rpm, 2 h) to form a homogeneous aqueous phase. Glycyrrhizic acid micelles were prepared using the same nanoparticle formulation procedure, but without the addition of the citriamide solution. In this embodiment, a Malvern laser particle size analyzer was used to determine the particle size of the nanoparticles using dynamic light scattering, and the zeta potential was determined using electrophoretic light scattering. Encapsulation efficiency and drug loading were determined using ultrafiltration centrifugation.

[0053] 1) Optimization of the mass ratio of glycyrrhizic acid to thiazolidinyl ether: With the total mass of GA (glycyrrhizic acid) and PAA (thiazolidinyl ether) fixed at 0.2% of the total mass of the aqueous dispersion system, the ethanol volume ratio at 8%, and the ultrasonic power at 220W, the mass ratios of glycyrrhizic acid to thiazolidinyl ether were set to 3:1, 5:1, 7:1, 11:1, and 13:1. The particle size, potential, encapsulation efficiency, and loading rate of the obtained self-assembled micelles were measured.

[0054] Table 1: Effect of glycyrrhizic acid:piperamide mass ratio on the product

[0055] The results showed that the micelle size was smallest (83.30±2.39 nm) when the mass ratio was 5:1. (See [reference needed]) Figure 3 It had the highest encapsulation efficiency (96.6±1.8%) and drug loading rate (10.97±1.61%), and the largest absolute value of Zeta potential (37.33±0.46mV).

[0056] 2) Optimization of ethanol volume fraction: With the glycyrrhizic acid:piperamide mass ratio fixed at 5:1 and the ultrasonic power at 220W, the ethanol volume fractions were set to 0%, 4%, 8%, 12%, and 16%.

[0057] Table 2: Effect of ethanol volume fraction on the product

[0058] The results of the product analysis showed that when the volume fraction of ethanol was 8%, the micelle size was the smallest (89.79±0.53nm), and the encapsulation efficiency (94.20±1.7%) and drug loading rate (11.76±0.34%) were the highest.

[0059] 3) Ultrasonic power optimization: With a fixed mass ratio of 5:1 and an ethanol volume ratio of 8%, set the ultrasonic power to 180W, 200W, 220W, 240W, and 260W.

[0060] Table 3 Effect of ultrasonic power on products

[0061] The results showed that when the ultrasonic power was 220W, the micelle size was the smallest (88.31±0.50nm), and the encapsulation efficiency (93.77±0.22%) and drug loading rate (11.69±1.78%) were the highest.

[0062] Therefore, the optimal preparation process was determined to be: glycyrrhizic acid to citric acid amide mass ratio of 5:1, ethanol volume fraction of 8%, and ultrasonic power of 220W.

[0063] Example 5

[0064] This embodiment provides a self-assembled glycyrrhizic acid nanoformulation loaded with zanthoxylate, comprising the following steps: (1) Using Sichuan pepper fruit peel powder as raw material, Sichuan pepper oil resin was obtained by supercritical CO2 extraction. The specific process is the same as in Example 1. (2) Dissolve the pepper oil resin in an ethanol aqueous solution, add macroporous weak acid cation exchange resin for decolorization treatment, filter, evaporate under reduced pressure to dryness, and obtain the decolorized product. (3) The decolorized product was loaded onto a polystyrene weakly polar adsorption resin column, eluted with methanol, the eluent was collected, and evaporated under reduced pressure to obtain crude zineb amide; steps (2) and (3) were performed in the same manner as in Example 2. (4) Dissolve the crude zanthoxylamide in methanol, separate it by preparative high performance liquid chromatography, collect the main peak fraction, evaporate it under reduced pressure and freeze dry to obtain zanthoxylamide; the specific operation is the same as in Example 3; (5) Preparation of glycyrrhizic acid aqueous solution of self-assembled micelles: Dissolve citriamide in ethanol and add it dropwise to the glycyrrhizic acid aqueous solution. Disperse by ultrasonication, remove ethanol by rotary evaporation under reduced pressure, and filter to obtain self-assembled micelles loaded with citriamide. Specifically: 1) Weigh an appropriate amount of glycyrrhizic acid and stir it in distilled water at 50℃ (500 rpm, 2 h) to form a homogeneous aqueous phase; 2) Dissolve the zanthoxylate prepared in Example 3 in an appropriate amount of ethanol aqueous solution, wherein the volume fraction of ethanol in the ethanol aqueous solution is 8%, and the mass ratio of zanthoxylate to glycyrrhizic acid is controlled to be 1:5; rapidly add the zanthoxylate ethanol solution dropwise into the glycyrrhizic acid aqueous solution, and continue stirring for 1 h; 3) After cooling to room temperature, use an ultrasonic cell disruptor to sonicate in an ice-water bath for 30 min (power 220 W, 3 s on / off). 4) Remove ethanol by rotary evaporation at 40℃, add distilled water to make up to a total mass concentration of 0.2wt% for the nano-formulation, filter through a 0.45 μm microporous membrane to obtain the glycyrrhizic acid self-assembled nano-formulation loaded with zanthoxylate.

[0065] Test case The following performance tests were performed on the zanthoxylate nanoformulation prepared in Example 5: (1) The particle size distribution of the nano-formulation was determined by dynamic light scattering method, and the results are as follows: Figure 4 As shown, the hydrated diameter of the nano-formulation is 83.89±1.46 nm, and the polydispersity index is 0.22±0.01. The morphology of the nano-formulation was observed using transmission electron microscopy, and the results are as follows. Figure 5 As shown, the nano-formulation is a regular spherical shape with a core-shell structure, a particle size of 50–80 nm (dry state), and is uniformly dispersed. (2) The intermolecular interactions of glycyrrhizic acid self-assembled nanoparticles loaded with cinnamamide were determined using Fourier transform infrared spectroscopy. GA, PAA, and PAA@GA nanoparticles were freeze-dried and then compressed with KBr into tablets, which were then heated at 4000–4000 cm⁻¹. - ¹ Scan within the range. Results are as follows: Figure 5 As shown, the amide I band of PAA in the PAA@GA nanoformulation starts from 1622 cm⁻¹. - ¹Blue shifted to 1643cm - ¹, Amide II band (1550cm) - ¹) The PAA completely disappeared, while the OH absorption peak of GA did not broaden, indicating that PAA was encapsulated in the hydrophobic core of GA, and the hydrophobic interaction is the main driving force for the formation of nano-formulations.

[0066] (3) The crystal structure of the glycyrrhizic acid self-assembled nanoparticles loaded with zanthamide was determined using X-ray diffraction. The scanning conditions were 4–45° and 5° / min. The results are as follows: Figure 6 As shown, both PAA and GA raw materials exhibit sharp characteristic diffraction peaks, while the PAA@GA nano-formulation only shows a broad bulging peak around 15°, and the original crystalline peak disappears, indicating that PAA exists in an amorphous form inside the nano-formulation.

[0067] (4) Thermal stability test: Free cinnamic acid and the nano-formulation obtained in Example 5 were heated at 90℃ for 4 hours, and samples were taken at 0, 1, 2, 3, and 4 hours. The cinnamic acid content was determined by HPLC. The results are as follows: Figure 7 As shown, the retention rate of free citronella amide decreased to 21.39±1.53%, while the retention rate of citronella amide in the nano-formulation was 71.14±0.60%, which was 2.34 times higher than that of free citronella amide.

[0068] (5) UV stability test: Free citric acid and the nano-formulation obtained in Example 5 were irradiated under 0.4 W / m² UV light for 24 h, and samples were taken and measured every 2 h. The results are as follows: Figure 8 As shown, the retention rate of free cinnamic acid was only 13.62±1.52%, while the retention rate of cinnamic acid in the nano-formulation was 75.58±1.31%.

[0069] (6) Storage stability test: Free citric acid and the nano-formulation obtained in Example 5 were stored at 4°C in the dark, 4°C under light, 25°C in the dark, and 25°C under light for 30 days, respectively, and samples were taken periodically for testing. The results are as follows: Figure 9 As shown, after 30 days of storage at 4℃ in the dark, the retention rate of zanthamide in the nano-formulation was as high as 95.76±0.16%, while the free zanthamide was only 32.50±0.32%.

[0070] The above results indicate that the glycyrrhizic acid self-assembled nanoformulation loaded with zanthoxylate prepared in this invention significantly improves the thermal stability, photostability and storage stability of zanthoxylate.

[0071] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a self-assembled nanoparticle formulation loaded with zanthamide, characterized in that, Includes the following steps: (1) Using Sichuan pepper fruit peel powder as raw material, Sichuan pepper oil resin was obtained by supercritical CO2 extraction. (2) Dissolve the pepper oil resin in an ethanol aqueous solution, add macroporous weak acid cation exchange resin for decolorization treatment, filter, evaporate under reduced pressure to dryness, and obtain the decolorized product. (3) The decolorized product was loaded onto a polystyrene weakly polar adsorption resin column, eluted with methanol, the eluent was collected, and evaporated under reduced pressure to obtain crude zanthamide. (4) Dissolve the crude zanthoxylamide in methanol, separate it by preparative high performance liquid chromatography, collect the main peak fraction, evaporate it under reduced pressure and freeze dry to obtain zanthoxylamide; (5) Glycyrrhizic acid was added to water to prepare an aqueous solution of glycyrrhizic acid. After dissolving citric acid in ethanol, the solution was added dropwise to the aqueous solution of glycyrrhizic acid. The solution was then dispersed by ultrasonication, the ethanol was removed by rotary evaporation under reduced pressure, and the solution was filtered to obtain a self-assembled nano-formulation loaded with citric acid.

2. The method for preparing self-assembled nano-formulations loaded with zanthamide according to claim 1, characterized in that, The conditions for supercritical CO2 extraction in step (1) are: extraction temperature 45~55℃, extraction pressure 20~30MPa, separation vessel I temperature 40~50℃, pressure 8~10MPa, separation vessel II pressure 3~5MPa, and extraction time 60~100min.

3. The method for preparing self-assembled nano-formulations loaded with zanthamide according to claim 1, characterized in that, The volume concentration of the ethanol aqueous solution in step (2) is 60-80%; the mass ratio of macroporous weak acid cation exchange resin to pepper oil resin is 1.5-2.5:1; the decolorization conditions are: temperature 25-35℃, oscillation speed 100-200r / min, and time 8-16h.

4. The method for preparing self-assembled nanoparticles loaded with zanthamide according to claim 1, characterized in that, The conditions for preparative high performance liquid chromatography described in step (4) are: C18 column, particle size 10 μm, inner diameter 10 mm, column length 250 mm; mobile phase 40~50% methanol-water; flow rate 2~4 mL / min.

5. The method for preparing self-assembled nanoparticles loaded with zanthamide according to claim 1, characterized in that, In step (5), the concentration of the glycyrrhizic acid aqueous solution is 1~5 mg / mL.

6. The method for preparing self-assembled nano-formulations loaded with zanthoxylate according to any one of claims 1 to 5, characterized in that, In step (5), the mass ratio of glycyrrhizic acid to citric acid amide is 3~13:1; the volume fraction of ethanol is 4~12%; the power of ultrasonic dispersion is 180~260W, and the time is 20~40min.

7. The method for preparing self-assembled nanoparticles loaded with zanthamide according to claim 6, characterized in that, In step (5), glycyrrhizic acid is added to water and heated to 40~60℃ to dissolve it, and the mass ratio of glycyrrhizic acid to citric acid amide is 5:

1.

8. The method for preparing self-assembled nano-formulations loaded with zanthamide according to claim 6, characterized in that, The volume fraction of ethanol is 8%; the power of ultrasonic dispersion is 220W.

9. The self-assembled nanoformulation loaded with citriamide obtained by the preparation method according to any one of claims 1 to 8.

10. The use of the self-assembled nanoformulation loaded with cinnamamide as described in claim 9 in the preparation of anti-inflammatory, antioxidant, or cell migration-promoting drugs or cosmetics.

Images