Process for preparing hyaluronic acid-paeonol-nicotinamide nanoparticles by using supercritical technology

Abstract

The invention provides a process for preparing hyaluronic acid-paeonol-nicotinamide nanoparticles by using a supercritical fluid technology, and belongs to the technical field of cosmetics. The method comprises the following steps: obtaining a micelle solution containing hyaluronic acid and water as a wall material; obtaining a core material containing paeonol and nicotinamide; mixing the wall material, the core material and the supercritical fluid solvent to obtain a supercritical preform; under a pre-expansion condition, mixing and stirring the supercritical prefabricate to form a supercritical microemulsion; and expanding the supercritical microemulsion to obtain the nanoparticles. The hyaluronic acid-paeonol-nicotinamide nanoparticles obtained by the invention have the characteristics of narrow particle size distribution and uniform size, and when the hyaluronic acid-paeonol-nicotinamide nanoparticles are applied to cosmetics, the transdermal ability, the skin wrinkle aging resistance, the skin tissue repair promotion, the oxidation resistance, the skin elasticity maintenance and the whitening effect of the hyaluronic acid-paeonol-nicotinamide nanoparticles are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of cosmetic technology, and in particular to a process for preparing hyaluronic acid-paeonol-nicotinamide nanoparticles by utilizing supercritical fluid technology and its application. Background Art

[0002] Skin whitening is one of the most important goals in people's pursuit of beauty. Therefore, cosmetics with whitening properties have become a hot topic in the cosmetics industry. The actual color of human skin is primarily determined by the content and distribution of melanin. If measures can be taken to reduce the deposition of melanin from melanocytes into keratinocytes, skin whitening can be effectively achieved.

[0003] Hyaluronic acid (HA) is a N Anionic acidic mucopolysaccharides composed of acetylglucosamine have excellent biocompatibility and moisturizing properties. As macromolecular self-assembled colloidal particles, they are highly promising biopharmaceutical materials. However, their strong hydrophilicity and poor surface activity make it difficult to prepare stable emulsions, limiting their development and application.

[0004] Niacinamide is currently the most widely used chemical whitening agent. Chinese invention patent CN117942278A discloses niacinamide-loaded nanoparticles using amphiphilic carboxymethylated β-glucan as a carrier material. These nanoparticles, with a particle size of 100-200 nm, exhibit excellent sustained-release, whitening, moisturizing, anti-inflammatory, and oil-control effects, demonstrating the advantages of nanoparticles in skin care. However, this method utilizes traditional emulsification methods to modify the β-glucan, making preparation relatively complex. Chinese invention patent CN110251419A discloses a facial mask essence containing paeonol as an active ingredient, which exhibits certain whitening and repairing effects. However, conventional emulsions have significant limitations in terms of absorption and efficacy. Therefore, the development of new niacinamide-paeonol-loaded nanoparticles and methods is of great significance. Summary of the Invention

[0005] In order to solve the technical problems existing in the background technology, the present invention proposes a preparation process of hyaluronic acid-paeonol-niacinamide nanoparticles, which combines supercritical coupling technology with supercritical fluid rapid expansion crystallization technology. Hyaluronic acid, paeonol and niacinamide are first mixed through supercritical coupling technology to form a supercritical microemulsion, and then directly formed into nanoparticles through supercritical fluid rapid expansion technology, thereby achieving efficient preparation of hyaluronic acid-paeonol-niacinamide nanoparticles and significantly improving the anti-skin aging effect.

[0006] In the present invention, the solvent of the supercritical fluid is carbon dioxide in a supercritical fluid state; the low-lipid-soluble ingredients hyaluronic acid, paeonol, and niacinamide can be dissolved in supercritical carbon dioxide in a supercritical state, or dissolved in supercritical fluid carbon dioxide to which a certain amount of co-surfactant is added; the carboxylate groups (-COO⁻) of hyaluronic acid and the amino groups (-NH2) of niacinamide are pre-assembled through electrostatic interaction, while the benzene rings of paeonol and the hydrophobic domains of hyaluronic acid are embedded through π-π stacking, thereby achieving spontaneous directional arrangement of the ternary complex; the prepared hyaluronic acid-paeonol-niacinamide nanoparticles have the characteristics of narrow particle size distribution and uniform size, and the hyaluronic acid-paeonol-niacinamide nanoparticles are used in cosmetics.

[0007] The purpose of the present invention is achieved through the following technical solutions: The present invention provides a process for preparing hyaluronic acid-paeonol-nicotinamide nanoparticles using supercritical technology, comprising the following steps: (1) Obtaining a micelle solution containing hyaluronic acid and water as a wall material; (2) obtaining a core material containing paeonol and niacinamide; (3) Mixing the wall material, the core material and the supercritical fluid solvent under sealed and supercritical conditions to obtain a supercritical preform; (4) Under pre-expansion conditions, the supercritical preform is mixed and stirred to form a supercritical microemulsion; (5) Nanoparticles are obtained by supercritical microemulsion expansion.

[0008] Furthermore, the supercritical fluid solvent is carbon dioxide in a supercritical fluid state.

[0009] Furthermore, the supercritical fluid solvent also contains a co-surfactant.

[0010] Furthermore, the cosurfactant is Tween 80.

[0011] Furthermore, the process is carried out in a supercritical reactor; First, the wall material, core material and co-surfactant are mixed and added into a supercritical reactor, and the lid is sealed; Then, carbon dioxide gas is introduced to replace the air in the supercritical reactor; The outlet valve of the supercritical reactor is opened, and the fluid is injected into the nanoparticle collector through the nanonozzle to obtain the hyaluronic acid-paeonol-nicotinamide nanoparticles.

[0012] After the fluid of the present invention is atomized by a nano nozzle with a certain aperture, it is rapidly precipitated and forms nuclei and crystals at the top of a nano particle collector due to a certain pressure drop, and then filtered and collected at the bottom of the nano collector to obtain hyaluronic acid-paeonol-nicotinamide nanoparticles.

[0013] Furthermore, when no co-surfactant is added, the mass ratio of the hyaluronic acid, paeonol, and niacinamide is 1:(0.2~0.8):(0.3~1.5); when a co-surfactant is added, the mass ratio of the hyaluronic acid, paeonol, niacinamide, and co-surfactant is (0.2~0.8):(0.3~1.5):(0.1~0.4).

[0014] Furthermore, the pre-expansion pressure is 10-30 MPa.

[0015] Furthermore, the pre-expansion temperature is 310 K to 350 K.

[0016] Furthermore, in step (4), the stirring speed is 200-500 rpm / min.

[0017] Furthermore, in step (4), the stirring time is 10 to 30 minutes.

[0018] Furthermore, the flow rate of the fluid ejected from the nano nozzle is 5-20 L / min.

[0019] Furthermore, the nozzle aperture of the nanonozzle is 1-100 nm.

[0020] Furthermore, the internal pressure of the nanoparticle collector is 3-5 MPa.

[0021] The present invention also provides hyaluronic acid-paeonol-nicotinamide nanoparticles prepared by the above process, wherein the particle size of the hyaluronic acid-paeonol-nicotinamide nanoparticles is 200-500 nm.

[0022] The present invention also provides an application of the above-mentioned hyaluronic acid-paeonol-nicotinamide nanoparticles in cosmetics, especially in cosmetics with the effects of increasing skin transdermal ability, resisting skin wrinkles and aging, promoting skin tissue repair, anti-oxidation, maintaining skin elasticity, and whitening.

[0023] The present invention also provides a cosmetic composition comprising the hyaluronic acid-paeonol-nicotinamide nanoparticles.

[0024] Furthermore, the cosmetic composition also includes other cosmetic active ingredients and excipients.

[0025] The cosmetics described in the present invention include facial cleansers, sunscreens, facial creams, and the like.

[0026] This invention combines supercritical coupling technology with supercritical fluid rapid expansion crystallization technology to achieve efficient preparation of hyaluronic acid-paeonol-niacinamide nanoparticles. The resulting hyaluronic acid-paeonol-niacinamide nanoparticles have a narrow and uniform particle size distribution. When used in cosmetics, hyaluronic acid-paeonol-niacinamide nanoparticles significantly enhance their transdermal penetration, anti-wrinkle and aging effects, skin tissue repair, antioxidant properties, skin elasticity maintenance, and whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 To evaluate the transdermal ability of hyaluronic acid-paeonol-niacinamide nanoparticles; Figure 2 To evaluate surface skin wrinkles in mice; Figure 3 HE staining analysis of mouse skin; Figure 4 To measure the SOD activity in mouse skin; Figure 5 To determine the content of MMP-3 in mouse skin; Figure 6 To determine the melanin content in mouse skin. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0029] The technical solution of the present invention is further described below with reference to specific embodiments.

[0030] The transdermal ability of the present invention is evaluated by the concentrations of niacinamide and paeonol in the Franz diffusion cell; the anti-wrinkle and aging effect is evaluated by the skin wrinkle score; the promotion of skin tissue repair is evaluated by HE staining sections; the anti-oxidation effect is evaluated by SOD activity; the maintenance of skin elasticity is evaluated by MMP-3 content; and the whitening effect is evaluated by melanin content.

[0031] Example 1: Preparation of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles Using Supercritical Fluid Technology 5 g of hyaluronic acid (HA) was added to 200 mL of deionized water and stirred to dissolve to obtain a micellar solution, which was then transferred to a supercritical carbon dioxide fluid reactor. 2 g of paeonol and 4 g of niacinamide were mixed and added to the reactor. 1 g of Tween 80 was added and the reactor capped. The carbon dioxide cylinder was opened and pressurized using a high-pressure pump before being injected into the supercritical carbon dioxide fluid reactor, displacing the air three times. The pre-expansion pressure was 15 MPa and the pre-expansion temperature was 320 K. The magnetic stirring speed was 200 r / min and the stirring time was 10 min until a microemulsion was fully formed. The outlet valve of the supercritical reactor was then opened, and the microemulsion flowed out of a nanonozzle with a 5 nm nozzle aperture at a flow rate of 5 L / min into a collection vessel. The pressure in the collection vessel was set at 5 MPa, and HA-paeonol-niacinamide nanoparticles with a particle size of approximately 300 nm were collected.

[0032] Example 2: Preparation of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles Using Supercritical Technology 5 g of hyaluronic acid (HA) was added to 200 mL of deionized water and stirred to dissolve to obtain a micellar solution, which was then transferred to a supercritical carbon dioxide fluid reactor. 2 g of paeonol and 4 g of niacinamide were mixed and added to the reactor. 1 g of Tween 80 was added and the reactor capped. The carbon dioxide cylinder was opened and pressurized using a high-pressure pump before being injected into the supercritical carbon dioxide fluid reactor. The air was displaced three times. The reactor was pressurized to a pre-expansion pressure of 15 MPa and a pre-expansion temperature of 320 K. The magnetic stirring speed was set at 200 r / min for 20 minutes until a microemulsion was fully formed. The outlet valve of the supercritical reactor was then opened, and the microemulsion flowed out of a nanonozzle with a 5 nm nozzle aperture at a flow rate of 5 L / min into a collection vessel. The pressure in the collection vessel was set at 5 MPa, and HA-paeonol-niacinamide nanoparticles with a particle size of approximately 350 nm were collected.

[0033] Example 3: Preparation of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles Using Supercritical Technology 5 g of hyaluronic acid (HA) was added to 200 mL of deionized water and stirred to dissolve to obtain a micellar solution, which was then transferred to a supercritical carbon dioxide fluid reactor. 2 g of paeonol and 4 g of nicotinamide were mixed and added to the reactor. 1 g of Tween 80 was added and the reactor capped. The carbon dioxide cylinder was opened and pressurized using a high-pressure pump before being injected into the supercritical carbon dioxide fluid reactor. The air was displaced three times. The reactor was pressurized to a pre-expansion pressure of 20 MPa and a pre-expansion temperature of 350 K. The magnetic stirring speed was set at 200 r / min for 20 minutes until a microemulsion was fully formed. The outlet valve of the supercritical reactor was then opened, and the microemulsion flowed out of a nanonozzle with a 5 nm nozzle aperture at a flow rate of 5 L / min into a collection vessel. The pressure in the collection vessel was set at 5 MPa, and hyaluronic acid-paeonol-nicotinamide nanoparticles with a particle size of approximately 280 nm were collected.

[0034] Example 4: Preparation of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles Using Supercritical Technology 5 g of hyaluronic acid (HA) was added to 200 mL of deionized water and stirred to dissolve to obtain a micellar solution, which was then transferred to a supercritical CO2 fluid reactor. 2 g of paeonol and 4 g of niacinamide were mixed and added to the reactor. 1 g of Tween 80 was added and the reactor capped. The CO2 cylinder was opened and pressurized using a high-pressure pump before being injected into the supercritical CO2 fluid reactor. The air was displaced three times. The reactor was pressurized to a pre-expansion pressure of 20 MPa and a pre-expansion temperature of 350 K. The solution was stirred magnetically at a speed of 300 r / min for 30 minutes until a microemulsion was fully formed. The outlet valve of the supercritical reactor was then opened, and the microemulsion flowed out of a nanonozzle with a 20 nm nozzle aperture at a flow rate of 2 L / min into a collection vessel. The pressure in the collection vessel was set at 10 MPa, and HA-paeonol-niacinamide nanoparticles with a particle size of approximately 420 nm were collected.

[0035] Example 5: Evaluation of the transdermal ability of hyaluronic acid-paeonol-nicotinamide nanoparticles The nanoparticles prepared in Example 3 were selected for the experiment. An experimental group (hyaluronic acid-paeonol-nicotinamide nanoparticles) and a control group (a mixture of sodium hyaluronate, niacinamide, paeonol collagen, and PDRN (mass ratio of 5:4:2:1)) were set up. The transdermal permeation test of the experimental group and the control group was performed using a Franz diffusion cell, and the nicotinamide and paeonol contents of the receiving solutions at different times were determined.

[0036] Nicotinamide content was determined using a nicotinamide assay kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.): Standard wells, blank wells, and sample wells were set up. 50 µL of standard of varying concentrations was added to each standard well, while no standard was added to the blank wells. 50 µL of the sample to be tested was added to the sample wells. 100 µL of horseradish peroxidase-labeled detection antigen was added to the standard and sample wells, excluding the blank wells. After incubation at 37°C for 60 minutes, the wells were washed five times with wash buffer. Subsequently, substrates A and B were thoroughly mixed at a 1:1 ratio by volume, and 100 µL of the substrate mixture was added to all wells. After incubation at 37°C for 15 minutes, 50 µL of stop solution was added to all wells, gently mixed, and absorbance was measured at 450 nm within 5 minutes using a preheated microplate reader.

[0037] Paeonol content was determined by liquid chromatography-mass spectrometry (LC-MS). An Agilent 1260 series LC system was used, using a Waters ACQUIT UPLC® BEH C18 (2.1 × 50 mm, 1.7 µm) column. The LC conditions were as follows: Phase A: 0.1% formic acid in water, Phase B: acetonitrile, Mobile phase gradient elution: 0–2 min, 10% Phase B; 2–5 min, 10%–90% Phase B; 5–7 min, 90% Phase B; 7–8 min, 90%–10% Phase B; 8–10 min, 10% Phase B, Flow rate: 0.3 mL / min, Column temperature: 35°C, Injection volume: 5 µL. The mass spectrometer was an AB SCIEX 4500 series LC system, operating in multiple reaction monitoring (MRM) mode with an electrospray ionization (ESI) source and scanning in negative ion mode. ESI source parameters: electrospray (IS) voltage -3000 V, curtain gas (CUR) pressure 35 Psi, nebulizer gas (GS1) pressure 50 Psi, auxiliary heater gas (GS2) pressure 50 Psi, temperature 500 °C, monitoring paeonol [MH]⁻, m / z 165.1 → 121.1.

[0038] The results are as follows Figure 1 As shown in Figure 2, at 8 h, the contents of nicotinamide and paeonol in the experimental group were 400.52 ± 21.31 μg / cm2, respectively, compared with those in the control group. 2 and 78.81 ± 7.91 µg / cm 2 , which were higher than those in the control group, indicating that hyaluronic acid-paeonol-nicotinamide nanoparticles have stronger transdermal ability.

[0039] Example 4: Evaluation of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles for Anti-Wrinkle Effect Nine SPF-grade ICR female mice weighing approximately 20 g were housed in strict accordance with animal handling guidelines and ethical requirements for animal experimentation, with three mice per cage. During the experiment, the mice were housed in an ambient temperature of 22 ± 2°C, a humidity of 55 ± 5%, and a 12-hour light-dark cycle. They were given free access to food. After one week of adaptive feeding, the mice were randomly divided into three groups. The remaining six mice, excluding the three mice in the control group, had their backs shaved (approximately 2 cm x 3 cm) using a shaver and depilatory cream. The mice were then randomly divided equally into two groups: a model group and a treatment group (using the hyaluronic acid-paeonol-nicotinamide nanoparticles prepared in Example 3). Mice in the treatment group had an appropriate amount of hyaluronic acid-paeonol-nicotinamide nanoparticles applied to the exposed skin area, allowing complete absorption. The mice were then exposed to combined UVA / UVB irradiation for 30 minutes. Mice in the model group were directly exposed to combined UVA / UVB irradiation for 30 minutes. Irradiation was performed once daily for 21 consecutive days. After the last UV combined irradiation, the skin condition of the mouse's back skin was recorded with a camera, and the wrinkle degree of the mouse skin was scored according to the wrinkle grade score (Table 1).

[0040] Table 1 Wrinkle grade score

[0041] By the results Figure 2 As can be seen from Table 2, the wrinkle grade score of the mice skin in the treatment group was 1.33 ± 0.58, which was significantly lower than that of the model group (4.67 ± 0.58), indicating that hyaluronic acid-paeonol-nicotinamide nanoparticles have the effect of alleviating skin aging and resisting skin wrinkles. ### P<0.001, *** P<0.001 was considered to be extremely significant.

[0042] Table 2 Skin wrinkle grade score table of mice in each group

[0043] Example 5: Evaluation of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles in Promoting Skin Tissue Repair A blank group, a model group, and a drug-treated group were set up respectively. The mice in the drug-treated group were smeared with the hyaluronic acid-paeonol-nicotinamide nanoparticles prepared in Example 3 on the exposed skin area and completely absorbed, and then subjected to UVA / UVB combined ultraviolet irradiation for 30 minutes; the mice in the model group were directly subjected to combined ultraviolet irradiation for 30 minutes; once a day, after continuous irradiation for 21 days, the mice were euthanized, and the skin tissue of the irradiated area was taken for subsequent research.

[0044] Mouse epidermis was fixed in 4% paraffin fixative for at least 24 hours. The trimmed tissue and corresponding labels were placed in a dehydration box for dehydration with graded alcohols and the paraffin blocks were melted. The melted wax was placed in an embedding frame and cooled in a -20°C paraffin embedding machine. The paraffin blocks were trimmed and sliced ​​on a paraffin microtome to a thickness of 4 μm. After drying the wax in water, the sections were removed and stored at room temperature until further use. Paraffin sections were then dewaxed to water, stained with hematoxylin and eosin, dehydrated, and mounted. Microscopic examination, image acquisition, and analysis were performed.

[0045] By the results Figure 3 The skin of the model mice showed typical signs of photoaging, characterized by abnormal epidermal thickness, disorganized and unevenly distributed dermal fibers, and disordered arrangement and abnormal shape of secretory cells and pores. The skin of the blank group and mice treated with the nanoparticles of the present invention had intact epidermal structure, normal thickness, clear cell arrangement, and normal morphology. The fibrous tissue in the dermis was orderly arranged and wavy. Therefore, it can be concluded that hyaluronic acid-paeonol-nicotinamide nanoparticles have the potential to protect and repair skin tissue.

[0046] Example 6: Evaluation of the Antioxidant Capacity of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles A blank group, a model group, and a treatment group were set up respectively. The hyaluronic acid-paeonol-nicotinamide nanoparticles prepared in Example 3 were applied to the exposed skin area of ​​the mice in the treatment group and completely absorbed, and then subjected to UVA / UVB combined ultraviolet irradiation for 30 minutes; the mice in the model group were directly subjected to combined ultraviolet irradiation for 30 minutes; once a day, after continuous irradiation for 21 days, the mice were euthanized, and the skin tissue of the irradiated area was taken for subsequent research.

[0047] Skin tissue was thawed at room temperature and homogenized in an ice bath. The mixture was then centrifuged at 8000 g for 10 minutes at 4°C. The supernatant was collected and placed on ice for analysis. Following the protocol of the SOD kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.), sample wells, blank wells, and control wells were set up. Sample wells were filled with 20 µL of reagent 1, 20 µL of the test sample, 70 µL of the extract, and 90 µL of the working solution. Blank wells were filled with 20 µL of reagent 1, 90 µL of the extract, and 90 µL of the working solution. Control wells were filled with 110 µL of the extract and 90 µL of the working solution. After the reagents were added, the mixture was mixed thoroughly and incubated at 25°C in the dark for 30 minutes. The absorbance of each well was measured at 450 nm.

[0048] SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1 - inhibition percentage) × V total] ÷ (W × V sample ÷ V sample total) =10×inhibition percentage ÷(1-inhibition percentage) ÷ W in, Inhibition percentage = (A 空白孔 - A 样品孔 ) ÷ (A 空白孔 - A 对照孔 ), A is the absorbance value V total: total volume of the reaction system, 0.2 mL W: sample mass, g V sample: the volume of sample added to the reaction system, 0.02 mL V total sample volume: total preparation volume of sample extract, 1 mL The results are as follows Figure 4 It can be seen that compared with the model group (9.38 ± 1.43 U / g), the SOD activity in the skin of mice in the treatment group was significantly increased (30.45 ± 6.58 U / g), indicating that hyaluronic acid-paeonol-nicotinamide nanoparticles can restore SOD activity in the skin and have potential anti-skin oxidation effects. "##" and "**" indicate P < 0.01, which is considered to be a very significant difference.

[0049] Example 7: Evaluation of Hyaluronic Acid-Paeonol-Nicotinamide Nanoparticles in Maintaining Skin Elasticity A blank group, a model group, and a treatment group were set up respectively. The hyaluronic acid-paeonol-nicotinamide nanoparticles prepared in Example 3 were applied to the exposed skin area of ​​the mice in the treatment group and completely absorbed, and then subjected to UVA / UVB combined ultraviolet irradiation for 30 minutes; the mice in the model group were directly subjected to combined ultraviolet irradiation for 30 minutes; once a day, after continuous irradiation for 21 days, the mice were euthanized, and the skin tissue of the irradiated area was taken for subsequent research.

[0050] Rinse the tissue with ice-cold PBS to remove residual blood, weigh it, and mince it. Thoroughly grind the minced tissue (g) to PBS containing 1 mM PMSF (mL) at a ratio of 1:9. Finally, centrifuge the homogenate at 5000 rpm / min for 10 minutes, and collect the supernatant for analysis. Following the instructions for the MMP-3 assay kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.), blank, standard, and sample wells were set up. Blank wells were left untreated. Standard wells were filled with 100 µL of various concentrations of standard solution. Sample wells were filled with 100 µL of pretreated sample. The plates were covered with adhesive tape and incubated at 37°C in the dark for 1.5 hours. After incubation, wash three times with 300 µL of 1x wash buffer per well. Then, add 100 µL of biotinylated antibody working solution to each well, mix gently, and incubate at 37°C in the dark for 1 hour. After incubation, wash four times using the same washing method. Next, add 100 µL of 1× SA-HRP working solution to each well. Incubate at 37°C in the dark for 30 min, wash four times, and pat dry. Next, add 50 µL of chromogenic solution A and 50 µL of chromogenic solution B to each well, mix gently, and incubate at 37°C in the dark for 15 min. After the color development reaction is complete, add 50 µL of stop solution to each well, mix gently, and measure the absorbance of each well at 450 nm.

[0051] By the results Figure 5 It can be seen that compared with the model group (407.77 ± 159.28 ng / g), the MMP-3 content in the skin of mice in the treatment group was significantly reduced (73.23 ± 11.98 ng / g), indicating that hyaluronic acid-paeonol-nicotinamide nanoparticles can significantly reduce the increase in MMP-3 content caused by ultraviolet irradiation, restore collagen and elastin levels, and have the potential to maintain skin elasticity. Among them, "##" and "**" indicate P < 0.01, which is considered to be a very significant difference.

[0052] Example 8: Evaluation of the whitening effect of hyaluronic acid-paeonol-nicotinamide nanoparticles A blank group, a model group, and a treatment group were set up respectively. The hyaluronic acid-paeonol-nicotinamide nanoparticles prepared in Example 3 were applied to the exposed skin area of ​​the mice in the treatment group and completely absorbed, and then subjected to UVA / UVB combined ultraviolet irradiation for 30 minutes; the mice in the model group were directly subjected to combined ultraviolet irradiation for 30 minutes; once a day, after continuous irradiation for 21 days, the mice were euthanized, and the skin tissue of the irradiated area was taken for subsequent research.

[0053] According to the experimental protocol of the melanin assay kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.), standard wells, blank wells, and sample wells were set up. 50 µL of standard of varying concentrations was added to each standard well, while no standard was added to the blank wells. 50 µL of the sample to be tested was added to the sample wells. 100 µL of horseradish peroxidase (HRP)-labeled detection antigen was added to the standard and sample wells, excluding the blank wells, and the plates were incubated at 37°C for 60 minutes. Subsequently, each well was washed with wash buffer, and the plate was washed five times. After washing, substrates A and B were thoroughly mixed at a 1:1 ratio, and 100 µL of the substrate mixture was added to all wells. The plate was covered with sealing film and incubated at 37°C for 15 minutes. Then, 50 µL of stop solution was added to all wells, gently mixed, and absorbance was measured at 450 nm within 5 minutes using a preheated microplate reader.

[0054] The results are as follows Figure 6 It can be seen that compared with the model group (18.34 ± 0.73 mg / g), the melanin content in the skin of mice in the treatment group was significantly reduced (6.81 ± 1.64 mg / g), indicating that hyaluronic acid-paeonol-nicotinamide nanoparticles have the ability to prevent melanin deposition and skin whitening. Among them, "###" and "***" indicate P<0.001, which is considered to be extremely significant.

[0055] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A process for preparing hyaluronic acid-paeonol-nicotinamide nanoparticles using supercritical technology, characterized in that: The following steps are involved: (1) Obtaining a micelle solution containing hyaluronic acid and water as a wall material; (2) obtaining a core material containing paeonol and niacinamide; (3) Mixing the wall material, the core material and the supercritical fluid solvent under sealed and supercritical conditions to obtain a supercritical preform; (4) Under pre-expansion conditions, the supercritical preform is mixed and stirred to form a supercritical microemulsion; (5) Nanoparticles are obtained by supercritical microemulsion expansion.

2. The process according to claim 1, characterized in that The supercritical fluid solvent is carbon dioxide in a supercritical fluid state; Preferably, the supercritical fluid solvent further contains a co-surfactant; Preferably, the cosurfactant is Tween 80; Preferably, the process is carried out in a supercritical reactor: first, the wall material, core material and co-surfactant are mixed and then added to the supercritical reactor, which is sealed; then carbon dioxide gas is introduced to replace the air in the supercritical reactor; the outlet valve of the supercritical reactor is opened, and the fluid is injected into the nanoparticle collector through a nanonozzle to obtain the hyaluronic acid-paeonol-niacinamide nanoparticles.

3. The process according to claim 1 or 2, characterized in that When no cosurfactant is added, the mass ratio of the hyaluronic acid, paeonol, and niacinamide is 1:(0.2-0.8):(0.3-1.5); When a cosurfactant is added, the mass ratio of the hyaluronic acid, paeonol, niacinamide and the cosurfactant is 1:(0.2-0.8):(0.3-1.5):(0.1-0.4).

4. The process according to any one of claims 1 to 3, characterized in that The pre-expansion pressure is 10-30 MPa, and / or the pre-expansion temperature is 310 K-350 K.

5. The process according to any one of claims 1 to 4, characterized in that In step (4), the stirring speed is 200-500 rpm / min, and / or the stirring time is 10-30 min.

6. The process according to any one of claims 2 to 5, characterized in that The fluid ejected from the nano nozzle has a flow rate of 5 to 20 L / min, and / or the nozzle aperture of the nano nozzle is 1 to 100 nm.

7. The process according to any one of claims 2 to 6, characterized in that The internal pressure of the nanoparticle collector is 3-5 MPa.

8. Hyaluronic acid-paeonol-nicotinamide nanoparticles prepared by the process according to any one of claims 1 to 7, characterized in that: The particle size of the hyaluronic acid-paeonol-nicotinamide nanoparticles is 200-500 nm.

9. Use of the hyaluronic acid-paeonol-nicotinamide nanoparticles according to claim 8 in cosmetics, especially in cosmetics having the effects of increasing skin transdermal ability, resisting skin wrinkles and aging, promoting skin tissue repair, anti-oxidation, maintaining skin elasticity, and whitening.

10. A cosmetic composition, characterized in that The invention comprises the hyaluronic acid-paeonol-nicotinamide nanoparticles according to claim 8.

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