Glycyrrhizin inclusion complex hydrogel whitening microneedles, their preparation method and application

Abstract

This invention relates to a glycyrrhizin inclusion complex cocrystalline hydrogel whitening microneedle, its preparation method, and its application. The glycyrrhizin inclusion complex cocrystalline hydrogel whitening microneedle includes a needle tip and a base. The needle tip is prepared from an aqueous solution of glycyrrhizin-HP-β-CD inclusion complex and a hyaluronic acid derivative. The glycyrrhizin-HP-β-CD inclusion complex is obtained by encapsulating glycyrrhizin with hydroxypropyl-β-cyclodextrin. The hyaluronic acid derivative is composed of L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid. The glycyrrhizin inclusion complex cocrystalline hydrogel whitening microneedle of this invention significantly improves the solubility, stability, and drug loading of glycyrrhizin. Furthermore, by utilizing the synergistic effect of sustained release and enhanced penetration, it significantly improves the transdermal efficiency of glycyrrhizin and increases the 24-hour skin retention of the drug, thereby enhancing the whitening, antioxidant, and anti-inflammatory effects of the formulation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and relates to microneedle formulations, specifically to a glycyrrhizin inclusion complex eutectic hydrogel whitening microneedle, its preparation method, and its application. Background Technology

[0002] Glabridin (GLA) is the main active ingredient extracted from licorice root, possessing significant whitening, antioxidant, and anti-inflammatory effects. However, its poor water solubility (solubility <0.1 mg / mL), poor chemical stability (easily oxidized and degraded), and low skin permeability limit its clinical application. Currently, most commercially available products are ordinary creams (such as Shiseido's White Lucent series in Japan), with a transdermal absorption rate of less than 5%.

[0003] There is a lack of stable delivery systems for glycyrrhizin in the existing technology, and traditional inclusion technology cannot simultaneously solve the problems of solubility, stability and transdermal absorption of glycyrrhizin. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a delivery system for glycyrrhizin to improve the solubility, stability and transdermal absorption of glycyrrhizin, thereby enhancing its whitening, antioxidant and anti-inflammatory effects.

[0005] The technical solutions for achieving the above objectives include the following.

[0006] In the first aspect, this invention provides a glycyrrhizin inclusion complex eutectic hydrogel whitening microneedle, comprising a needle tip and a substrate, wherein the needle tip is prepared from an aqueous solution of glycyrrhizin-HP-β-CD inclusion complex and hyaluronic acid derivative;

[0007] The glycyrrhizin-HP-β-CD inclusion complex was obtained by encapsulating glycyrrhizin with hydroxypropyl-β-cyclodextrin;

[0008] The hyaluronic acid derivative is composed of L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid.

[0009] Secondly, the present invention provides a method for preparing the whitening microneedles of the glycyrrhizin inclusion complex eutectic hydrogel, comprising the following steps:

[0010] (1) Dissolve the glycyrrhizin-HP-β-CD inclusion complex in water to prepare a homogeneous aqueous solution, then add the L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid, mix evenly to obtain a needle tip solution;

[0011] (2) Add the needle tip solution to the microneedle negative mold, centrifuge to fill the microchannels of the negative mold with the needle tip solution, scrape off the needle tip solution on the surface of the microneedle negative mold, centrifuge again to fully compress the needle tip solution into the microchannels of the mold;

[0012] (3) Dry the microneedle negative mold containing the needle tip solution obtained in step (2);

[0013] (4) Take the base solution and spread it evenly on the microneedle negative mold after drying in step (3), centrifuge, dry, and take out the microneedle negative mold at certain intervals during the drying process, add base solution and centrifuge, so that the base solution completely covers the micropores;

[0014] (5) After adding the base solution for the last time and centrifuging, the microneedle negative mold is dried and demolded to obtain the whitening microneedle of the glycyrrhizin inclusion complex eutectic hydrogel.

[0015] Thirdly, the present invention provides the application of the L-serine-grafted hyaluronic acid or L-lysine-grafted hyaluronic acid or hyaluronic acid derivative or the glycyrrhizin inclusion complex eutectic hydrogel whitening microneedles in the preparation of whitening, antioxidant and / or anti-inflammatory products.

[0016] The present invention has the following beneficial effects:

[0017] This invention uses hydroxypropyl-β-cyclodextrin (HP-β-CD) to encapsulate glycyrrhizin, which significantly improves the solubility, stability and encapsulation rate of glycyrrhizin, and solves the problems of poor solubility and easy oxidation and degradation of traditional formulations.

[0018] This invention uses L-serine and L-lysine to graft and modify hyaluronic acid, respectively. The L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid are then used as a compound matrix to prepare a co-crystalline hydrogel microneedle containing glycyrrhizin-HP-β-CD inclusion complex. Through amino acid modification, not only is the biological function of hyaluronic acid enhanced, but the drug loading capacity of the microneedle for glycyrrhizin and the mechanical strength of the microneedle are also effectively increased, improving the transdermal delivery rate and thus effectively enhancing the whitening, antioxidant, and anti-inflammatory effects of the microneedle.

[0019] This invention encapsulates glycyrrhizin with hydroxypropyl-β-cyclodextrin (HP-β-CD) and combines this with amino acid modification of the hyaluronic acid microneedle matrix (eutectic hydrogel microneedle delivery system). The glycyrrhizin-HP-β-CD inclusion complex is loaded onto the microneedle tip, achieving hydrophilic matrix loading of a hydrophobic drug. Through the unique drug delivery method of microneedles, the transdermal absorption of glycyrrhizin is improved. The synergistic effect of these two components significantly improves the solubility, stability, and drug loading of glycyrrhizin. Furthermore, the sustained-release and penetration-enhancing synergistic effect significantly improves the transdermal efficiency of glycyrrhizin and increases the amount of drug retained in the skin. This invention, through three levels of innovation—molecular inclusion, material modification, and device design—solves the technical bottlenecks of low transdermal efficiency and short duration of effect in traditional whitening preparations, significantly improving the whitening, antioxidant, and anti-inflammatory effects of the formulation. Attached Figure Description

[0020] Figure 1 This is a standard curve for the determination of glycyrrhizin content.

[0021] Figure 2 XRD patterns of GLA, HP-β-CD, HP-β-CD and physical mixtures and inclusion complexes of GLA.

[0022] Figure 3 DSC spectra of GLA, HP-β-CD, HP-β-CD and physical mixtures and inclusion complexes of GLA.

[0023] Figure 4 The inhibitory effects of free GLA and HP-β-CD-GLA inclusion complex on tyrosinase activity at different time points were investigated.

[0024] Figure 5 This is the NMR spectrum of hyaluronic acid.

[0025] Figure 6 The NMR spectrum of L-serine grafted with hyaluronic acid (HA-Ser).

[0026] Figure 7 The NMR spectrum of L-lysine grafted with hyaluronic acid (HA-Lys).

[0027] Figure 8 Dynamic rheological properties of the eutectic hydrogel at frequencies of 1–100 rad / s.

[0028] Figure 9 Design parameters for microneedle male mold (unit: mm).

[0029] Figure 10 Scanning electron microscope image of whitening microneedles made from glabridin inclusion complex eutectic hydrogel.

[0030] Figure 11This is a graph representing the mechanical properties of the microneedles (stress-displacement curve).

[0031] Figure 12 The cumulative transdermal dose of whitening microneedles containing glaucoma compound eutectic hydrogel.

[0032] Figure 13 The skin retention amount of the whitening microneedles is determined by the inclusion complex eutectic hydrogel of glycyrrhizin.

[0033] Figure 14 The effect of different microneedle groups on the ABTS free radical scavenging rate.

[0034] Figure 15 ROS-stained fluorescence images of Hacat cells, a photoaging model, after incubation with different microneedle groups.

[0035] Figure 16 The results of SOD and MDA detection in Hacat cells, a photoaging model, after treatment with different microneedle groups are shown.

[0036] Figure 17 The expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 after treatment with different microneedles. Detailed Implementation

[0037] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0038] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0039] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0040] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0041] Some of the embodiments involve a glycyrrhizin inclusion complex eutectic hydrogel skin whitening microneedle, comprising a tip and a substrate, wherein the tip is prepared from an aqueous solution of glycyrrhizin-HP-β-CD inclusion complex and a hyaluronic acid derivative;

[0042] The glycyrrhizin-HP-β-CD inclusion complex was obtained by encapsulating glycyrrhizin with hydroxypropyl-β-cyclodextrin;

[0043] The hyaluronic acid derivative is composed of L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid.

[0044] In some embodiments, the mass ratio of the glycyrrhizin-HP-β-CD inclusion complex to the hyaluronic acid derivative is 1:1-3, more preferably 1:1.5-2.5, and even more preferably 1:1.8-2.2.

[0045] In some embodiments, the aqueous solution of the glycyrrhizin-HP-β-CD inclusion complex and the hyaluronic acid derivative contains 15%-25% by mass of the hyaluronic acid derivative, more preferably 18%-22%.

[0046] In some embodiments, the mass ratio of L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid in the hyaluronic acid derivative is 1:0.5-1.5, preferably 1:0.8-1.2.

[0047] In some embodiments, the L-serine-grafted hyaluronic acid is obtained by an amidation reaction of hyaluronic acid and L-serine, and the L-lysine-grafted hyaluronic acid is obtained by an amidation reaction of hyaluronic acid and L-lysine.

[0048] In some embodiments, the molecular weight of the hyaluronic acid is 300,000 to 1,000,000, preferably 400,000 to 800,000.

[0049] In some embodiments, the mass ratio of hyaluronic acid to L-serine is 1.5-2:1.

[0050] In some embodiments, the mass ratio of hyaluronic acid to L-lysine is 1-1.5:1.

[0051] In some embodiments, the preparation method of L-serine-grafted hyaluronic acid or L-lysine-grafted hyaluronic acid includes the following steps:

[0052] The hyaluronic acid is dissolved in a buffer solution, an activator is added to activate the carboxyl group of the hyaluronic acid, and then L-serine or L-lysine is added. The reaction is carried out to obtain L-serine-grafted hyaluronic acid or L-lysine-grafted hyaluronic acid.

[0053] In some of these embodiments, the pH of the buffer solution is 4.5-5.5.

[0054] In some of these embodiments, the buffer solution is an MES buffer solution.

[0055] In some embodiments, the activator is carbodiimide and N-hydroxysuccinimide, and the mass ratio of carbodiimide to N-hydroxysuccinimide is preferably 1.5-2:1.

[0056] In some embodiments, the amount of the activator is 0.9-1.2 times the weight of the hyaluronic acid.

[0057] In some of these embodiments, the activation temperature is 40°C-50°C and the time is 10 min-20 min.

[0058] In some of these embodiments, the reaction is carried out at a temperature of 40°C-50°C for a duration of 20-28 hours.

[0059] In some embodiments, the glycyrrhizin-HP-β-CD inclusion complex is prepared from glycyrrhizin and hydroxypropyl-β-cyclodextrin in a molar ratio of 1:1.2-1.8.

[0060] In some embodiments, the preparation method of the glycyrrhizin-HP-β-CD inclusion complex includes the following steps:

[0061] The glycyrrhizin and hydroxypropyl-β-cyclodextrin were prepared into ethanolic solutions of glycyrrhizin and aqueous solutions of hydroxypropyl-β-cyclodextrin with concentrations of 0.3 mol / L to 0.32 mol / L, respectively. The ethanolic solution of glycyrrhizin was slowly titrated into the aqueous solution of hydroxypropyl-β-cyclodextrin under constant temperature conditions of 35-45℃ until a transient white precipitate appeared, at which point the titration was stopped. The reaction system was stirred continuously for 20-28 hours, the ethanol was removed, the unencapsulated drug was removed by filtration, and the mixture was lyophilized to obtain the glycyrrhizin-HP-β-CD inclusion complex.

[0062] In some embodiments, the substrate is prepared from an aqueous solution of oligomeric hyaluronic acid.

[0063] In some embodiments, the oligomeric hyaluronic acid has a molecular weight of 20 kDa or less, preferably 10 kDa or less.

[0064] In some embodiments, the mass concentration of the oligomeric hyaluronic acid aqueous solution is 15%-25%, more preferably 18%-22%.

[0065] Some of the embodiments involve a method for preparing the whitening microneedles of the glycyrrhizin inclusion complex eutectic hydrogel, including the following steps:

[0066] (1) Dissolve the glycyrrhizin-HP-β-CD inclusion complex in water to prepare a homogeneous aqueous solution, then add the L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid, mix evenly to obtain a needle tip solution;

[0067] (2) Add the needle tip solution to the microneedle negative mold, centrifuge to fill the microchannels of the negative mold with the needle tip solution, scrape off the needle tip solution on the surface of the microneedle negative mold, centrifuge again to fully compress the needle tip solution into the microchannels of the mold;

[0068] (3) Dry the microneedle negative mold containing the needle tip solution obtained in step (2);

[0069] (4) Take the base solution and spread it evenly on the microneedle negative mold after drying in step (3), centrifuge, dry, and take out the microneedle negative mold at certain intervals during the drying process, add base solution and centrifuge, so that the base solution completely covers the micropores;

[0070] (5) After adding the base solution for the last time and centrifuging, the microneedle negative mold is dried and demolded to obtain the whitening microneedle of the glycyrrhizin inclusion complex eutectic hydrogel.

[0071] In some of these embodiments, the centrifugation conditions in step (2) include: 3500 rpm-4500 rpm, 4℃-10℃, 25min-35min.

[0072] In some of these implementations, step (2) is repeated 2-4 times.

[0073] In some of these embodiments, the drying temperature in step (3) is 15°C-40°C and the time is 20-28 hours.

[0074] In some of these embodiments, the centrifugation conditions in step (4) include: 3500 rpm-4500 rpm, 4℃-10℃, 8min-15min.

[0075] In some of these implementations, step (4) refers to adding the base solution and centrifuging 2-5 times every 3-5 hours.

[0076] In some of these embodiments, the drying temperature in step (4) is 15°C-40°C.

[0077] In some of these embodiments, the drying temperature in step (5) is 15°C-40°C and the time is 40-56 hours.

[0078] The present invention will be further described in detail below with reference to specific embodiments.

[0079] In this invention, glycyrrhizin is abbreviated as GLA;

[0080] Hydroxypropyl-β-cyclodextrin is abbreviated as HP-β-CD.

[0081] Example 1: Preparation and characterization of glycyrrhizin-HP-β-CD inclusion complex

[0082] 1.1 Preparation of glycyrrhizin-HP-β-CD inclusion complex (hereinafter referred to as HP-β-CD-GLA)

[0083] Phase solubility method: First, accurately prepare 0.31 mol / L GLA ethanol solution and HP-β-CD aqueous solution respectively. Under constant temperature of 40℃, slowly titrate the GLA ethanol solution into the HP-β-CD aqueous solution until the instantaneous white precipitate appears (at this point, the molar ratio of GLA to HP-β-CD is 1:1.5). After the reaction system is continuously stirred for 24 h, the ethanol is removed by rotary evaporation. After the filtrate is pre-frozen at -20 ℃ for 12 hours, it is freeze-dried in a freeze dryer (-55℃) for three days to obtain the final inclusion complex.

[0084] Coprecipitation method: Weigh appropriate amounts of GLA and HP-β-CD at a molar ratio of 1:1. Dissolve GLA in ethanol to prepare a 0.31 mol / L GLA solution; dissolve HP-β-CD in deionized water to prepare a 0.31 mol / L HP-β-CD solution. Add the GLA ethanol solution dropwise to the HP-β-CD solution. Stir at 40 °C for 24 hours, then remove the ethanol by rotary evaporation. Filter the solution (to obtain unincluded GLA). Pre-freeze the filtrate at -20 °C for 12 hours, and finally freeze-dry it in a freeze dryer (-55 °C) for three days to obtain the desired inclusion complex.

[0085] 1.2 Characterization of the glycyrrhizin-HP-β-CD inclusion complex

[0086] 1.2.1 Determination of glycyrrhizin content

[0087] Standard curve preparation: Accurately weigh 324.37 mg of glycyrrhizin (98% purity) and place it in a 10 mL amber volumetric flask. Dissolve and dilute to the mark with methanol, then shake well to obtain a stock solution of glycyrrhizin reference standard with a concentration of approximately 100 mM. Accurately measure an appropriate amount of this stock solution and serially dilute it with methanol in 10 mL volumetric flasks, diluting to the mark to prepare a series of glycyrrhizin reference standard solutions with concentrations of 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, and 500 μM. Filter the above series of solutions through a 0.22 μm microporous membrane and analyze them using a liquid chromatograph, recording the corresponding peak areas (y).

[0088] The glycyrrhizin was quantitatively analyzed using a high-performance liquid chromatography (HPLC) system, with the specific parameters as follows:

[0089]

[0090] like Figure 1 As shown: A standard curve was established under the above chromatographic conditions. The retention time of glycyrrhizin was 4.879 min, and the standard curve was: y = 13.776x - 28.734(R). ² = 0.999), indicating that glycyrrhizin has good linearity in the injection range of 0.1-500 μM.

[0091] 1.2 Encapsulation efficiency test

[0092] Two 5 mg samples of the glycyrrhizin-HP-β-CD inclusion complex were prepared. One sample was added to 10 mL of methanol and sonicated for 30 min at room temperature. An appropriate amount of the sample was then filtered through a 0.22 μm filter membrane for liquid chromatography analysis to determine the total glycyrrhizin content in the inclusion complex. The other sample was added to 10 mL of deionized water and sonicated for 30 min at room temperature. The solution was then subjected to liquid chromatography at 3000 rpm. -1 Centrifuge for 15 min, filter the supernatant through a 0.22 mm filter membrane, and perform high-performance liquid chromatography (HPLC) to determine the content of encapsulated glycyrrhizin. The experiment was repeated three times, and the average value was taken as the result. The encapsulation efficiency was calculated using the following formula:

[0093] Encapsulation efficiency = (Encapsulated glycyrrhizin content / Total glycyrrhizin content in the sample) × 100%

[0094] 1.3 Drug loading rate test

[0095] Take 5 mg of glycyrrhizin-HP-β-CD inclusion complex sample, add it to 10 mL of methanol, and sonicate for 30 min at room temperature. Filter an appropriate amount of the sample through a 0.22 μm filter membrane and perform high-performance liquid chromatography (HPLC) analysis to determine the total glycyrrhizin content in the inclusion complex. Repeat the experiment three times and take the average value as the analytical result. The drug loading rate is calculated according to the following formula:

[0096] Drug loading rate = (Glycyrrhizin content in the sample / total sample mass) × 100%

[0097] The encapsulation efficiency and drug loading rates obtained by the two methods were calculated, revealing that the encapsulation efficiency of the inclusion complex obtained by the phase solubility method reached 89.96±1.89%, and the drug loading rate was 15.98±0.89%; while the encapsulation efficiency of the inclusion complex obtained by the coprecipitation method was only 48.98±0.78%, and the drug loading rate was 14.34±0.56%. The phase solubility method significantly improved the encapsulation efficiency compared to the coprecipitation method. The phase solubility method, through slow titration to a defined precipitation endpoint, ensured that the drug addition was close to the maximum inclusion capacity (high inclusion rate) of HP-β-CD, thus obtaining a high-purity inclusion complex product with a high drug loading. Furthermore, compared to the coprecipitation method, the phase solubility method eliminates the need for filtration in product purification, making the operation simpler.

[0098] 1.4 X-ray diffraction test

[0099] Take a small amount of GLA, HP-β-CD, PM (a physical mixture of HP-β-CD and GLA), and HP-β-CD-GLA powder into an X-ray diffractometer. The test conditions are: scanning angle of 2Θ, scanning speed of 10°·min', scanning range of 2~50°, voltage of 40kV, and current of 15mA.

[0100] X-ray diffraction (XRD) analysis results (such as...) Figure 2 The active pharmaceutical ingredient (API) of glycyrrhizin (GLA) exhibited distinct crystalline diffraction peaks at characteristic diffraction angles of 8.5°, 16.2°, 17.8°, and 19.5°, consistent with reported crystal structure characteristics. The XRD pattern of the physical mixture (PM) showed a simple superposition of the characteristic peaks of glycyrrhizin and HP-β-CD, with the main diffraction peaks of glycyrrhizin at 16.2°, 17.8°, and 19.5° still observable. The XRD pattern of the inclusion complex showed typical amorphous diffuse peaks, with all crystalline diffraction peaks of the API completely disappearing, indicating that glycyrrhizin was successfully included by HP-β-CD, forming an amorphous complex with a higher energy state. This phase transition from crystalline to amorphous state is a key factor in the inclusion complex's significant improvement in the solubility of glycyrrhizin, as amorphous drugs have higher surface free energy and thermodynamic activity, which facilitates their rapid dissolution and release in aqueous media.

[0101] 1.5 Differential Scanning Calorimetry Experiment

[0102] Small amounts of GLA, HP-β-CD, PM (a physical mixture of HP-β-CD and GLA), and HP-β-CD-GLA powders were placed in a low-temperature differential scanning calorimeter. The test conditions were: reference empty aluminum crucible, N2 atmosphere, gas flow rate of 20 mL / min, and heating rate of 10 °C / min. -1 The scanning range is 25~300℃.

[0103] Differential scanning calorimetry (DSC) is a reliable and rapid thermal analysis method that measures the energy change between a sample and a reference material as a function of temperature. The DSC spectra of GLA, HP-β-CD, physical mixtures (PM), and inclusion complexes (HP-β-CD-GLA) are shown below. Figure 3 As shown in the figure. Test results indicate that glycyrrhizin exhibits a characteristic melting peak at approximately 230 °C; in the 125-175 °C range, HP-β-CD shows a broad endothermic melting peak, which is related to the moisture loss of HP-β-CD; HP-β-CD, the physical mixture, and the inclusion complex all show endothermic peaks indicating moisture evaporation around approximately 150 °C. In the DSC spectrum of the inclusion complex (HP-β-CD-GLA), no characteristic melting peak of GLA was observed, indicating that glycyrrhizin is uniformly dispersed in HP-β-CD in an amorphous form, a result consistent with XRD analysis.

[0104] 1.6 Determination of Tyrosinase Inhibitory Activity

[0105] Tyrosinase inhibitory activity was determined using L-DOPA as a substrate. The experiment began with the preparation of 0.5 mg / mL... -1 A high-concentration stock solution of GLA was prepared, and the prepared HP-β-CD-GLA inclusion complex was diluted with deionized water to a GLA concentration of 0.25 mg / mL. -1 The high-concentration solutions were then prepared, and the GLA stock solution and HP-β-CD-GLA inclusion complex solution were diluted with PBS to concentrations of 0.1, 0.5, 1, 2, 4, 6, and 8 μg / mL, respectively. -1 A series of concentrations were determined. Accurately weigh 1.97 mg L-DOPA, dissolve it in PBS, and dilute to a 10 mL volumetric flask. Simultaneously, prepare mushroom tyrosinase at a concentration of 100 U / mL. -1 The enzyme solution was prepared by mixing 1 mL of L-DOPA solution with 1 mL of sample solutions of different concentrations in a centrifuge tube. The mixture was incubated at 37 °C in the dark for 5 min, followed by the addition of 0.5 mL of mushroom tyrosinase solution. The reaction was continued at 37 °C for 10 min. After the reaction was complete, the absorbance was measured at 475 nm, and the inhibition rate of the sample on mushroom tyrosinase activity was calculated using the formula.

[0106] Tyrosinase inhibition rate % = (A 不加样品 -A 加样品 ) / A 不加样品 *100%

[0107] The results are as follows Figure 4 As shown, HP-β-CD-GLA exhibited an inhibition rate of 78.9%, comparable to that of free GLA. However, with prolonged storage, GLA gradually deteriorated, and its inhibitory activity against tyrosinase rapidly decreased, dropping to 45% after 3 hours. In contrast, HP-β-CD-GLA remained relatively stable, maintaining an inhibition rate of over 70% after 3 hours. This indicates that glycyrrhizin has been successfully encapsulated by HP-β-CD, and that HP-β-CD encapsulation significantly improves the stability of glycyrrhizin.

[0108] Example 2 Synthesis of amino acid-modified hyaluronic acid

[0109] This embodiment employs an amidation reaction mediated by carbodiimide (EDC) / N-hydroxysuccinimide (NHS) to modify hyaluronic acid (HA, molecular weight 400,000-800,000, purchased from Maclean's, catalog number: H909937) through amino acid grafting. The reaction formula and specific steps are as follows:

[0110]

[0111] Where y = nx.

[0112] 4.0 g of hyaluronic acid was dissolved in 400 mL of MES (2-morpholinoethanesulfonic acid) buffer (0.5 mol / L, pH 5.0). After dissolution by stirring at 45 °C, 2.4 g of EDC·HCl and 1.4 g of NHS were added sequentially to activate the carboxyl groups for 15 min. Then, 2.12 g of L-serine or 3.0 g of L-lysine was added, and the reaction was carried out at 45 °C for 24 h. The reaction product was purified by dialysis using an 8 kDa dialysis bag (dialysis with ultrapure water for 72 h, with buffer changes every 8 h), and lyophilized to obtain L-serine-grafted hyaluronic acid (HA-Ser) and L-lysine-grafted hyaluronic acid (HA-Lys), which were stored in a desiccator for later use.

[0113] The NMR spectra of hyaluronic acid, L-serine-grafted hyaluronic acid (HA-Ser), and L-lysine-grafted hyaluronic acid (HA-Lys) are as follows: Figures 5-7As shown in the HA-Ser spectrum, the newly appearing multiplet at δ 3.70–3.90 ppm corresponds to the L-serine β-methylene proton; similarly, the triplet at δ 2.90–3.10 ppm in the HA-Lys spectrum corresponds to the lysine ε-methylene proton; the relative integral area of ​​the acetyl matrix proton peak (δ 1.90 ppm) in both modified products is reduced, which is consistent with the expectation that some carboxyl groups participate in the grafting reaction. These structural changes not only demonstrate the success of the chemical modification, but more importantly, introduce new functional groups into hyaluronic acid, which will significantly enhance its potential for subsequent applications as a drug carrier, such as enhancing drug loading capacity and improving biocompatibility.

[0114] Example 3 Preparation of eutectic hydrogel

[0115] This embodiment uses a swelling method to prepare a hyaluronic acid derivative eutectic hydrogel. The specific steps are as follows:

[0116] Accurately weigh a certain amount of L-serine-grafted hyaluronic acid (HA-Ser) and L-lysine-grafted hyaluronic acid (HA-Lys), and prepare homogeneous solutions with a mass fraction of 10% using ultrapure water. Mix the resulting solutions 1:1 and place them in a 37℃ constant temperature shaking incubator, where they are continuously shaken at 100 rpm for 12 h to swell, finally obtaining a eutectic hydrogel.

[0117] To verify the formation of the eutectic hydrogel, its rheological characterization was performed using a rotational rheometer. Using a parallel plate fixture (plate spacing set to 1 mm), its viscoelastic behavior was determined by strain scanning within the linear viscoelastic region. Subsequently, frequency scanning (frequency range: 0.1–100 rad / s) was conducted, recording the changes in storage modulus (G′) and loss modulus (G″) as a function of frequency. All rheological experiments were performed at a constant temperature of 37°C, and each sample was measured three times to ensure the reliability of the results. Finally, the formation of the eutectic hydrogel was clearly determined by the numerical relationship and frequency dependence of G′ and G″.

[0118] Rheological experimental results as follows Figure 8 As shown, in the angular frequency range of 0.1 to 100 rad / s, the storage modulus (G') is higher than the loss modulus (G”), indicating that an elastic hydrogel with a three-dimensional cross-linked network has been formed.

[0119] Example 4: Preparation and characterization of glycyrrhizin inclusion complex eutectic hydrogel whitening microneedles

[0120] I. Preparation of whitening microneedles from glabridin inclusion complex eutectic hydrogel

[0121] 1. Preparation of microneedle positive mold

[0122] The microneedle body was designed with a height of 1200 μm (including a 600 μm long cuboid section and a 600 μm long triangular pyramid section), a center-to-center spacing of 800 μm, and a microneedle master mold containing 144 microneedles per microneedle array. Microneedle design drawings were created and converted into 2D and 3D models using computer-aided design (CAD) (e.g., ...). Figure 9 As shown in the figure, by combining micro-precision machining technology and precision CNC machine tools, micro-needle positive molds are prepared by fine machining using brass as raw material.

[0123] 2. Preparation of microneedle negative mold

[0124] After ultrasonically cleaning the positive mold with anhydrous ethanol, weigh out an appropriate amount of polydimethylsiloxane (PDMS) and curing agent, mix them evenly at a ratio of 10:1 (w / w), pour the mixture into the prepared positive mold, and remove air bubbles in a vacuum drying oven at 0.8 bar for 30 min. After the air bubbles are completely eliminated, place it in an 80 °C constant temperature oven for 1 h to cure. After cooling to room temperature, carefully peel it off to obtain the PDMS microneedle negative mold.

[0125] 3. Preparation of needle tip (or needle body) solution

[0126] The glycyrrhizin-HP-β-CD inclusion complex was dissolved in ultrapure water to prepare a homogeneous aqueous solution with a mass concentration of 10% (ultrasonic-assisted dissolution, 25℃, 200 W, 10 min). HA-Ser and HA-Lys powders were weighed and slowly added to the inclusion complex aqueous solution to make the final mass fractions of hyaluronic acid derivatives HA-Ser and HA-Lys 10% respectively. The mixture was then thoroughly mixed to obtain a needle tip solution.

[0127] 4. Preparation of the base solution

[0128] Oligomeric hyaluronic acid (molecular weight <10 kDa, purchased from Bio-Tech, catalog number: JY-A0516049) was dissolved in water to prepare a 20% aqueous solution, which was used as the base solution. Because oligomeric hyaluronic acid has a small molecular weight, it is easily soluble in skin exudate, facilitating the separation of the microneedle tip from the base.

[0129] 5. Preparation of skin whitening eutectic hydrogel microneedles

[0130] Microneedles were prepared using a multi-step centrifugal microinfusion method, the specific steps of which are as follows:

[0131] (1) Take the prepared needle tip solution and microneedle negative mold, use the syringe barrel part of a disposable syringe to take 200 μL of needle tip solution and place it on the negative mold. Place the negative mold in the basket of a basket centrifuge and centrifuge at 4000 rpm and 4-10 ℃ for 30 min to fill the micropores of the negative mold with needle tip solution.

[0132] (2) Remove the female mold, use an aluminum scraper to thoroughly scrape off the needle tip solution on the surface of the female mold, then place the female mold in a basket and centrifuge at 4000 rpm, 4-10℃ for 30 minutes to fully compress the needle tip solution into the micropores of the mold;

[0133] (3) Repeat steps (1) and (2) three times;

[0134] (4) Place the microneedle negative mold containing the needle tip solution in a desiccator and dry at room temperature for 24 hours;

[0135] (5) Take out the microneedle negative mold from the desiccator, take 500 μL of microneedle base solution and spread it evenly on the negative mold, centrifuge at 4000 rpm and 4-10 ℃ for 10 min, put it in the desiccator to dry at room temperature, take it out every 4 h to add base solution and centrifuge at 4000 rpm and 4-10 ℃ for 10 min to completely cover the micropores, repeat three times;

[0136] (6) After the last addition of liquid and centrifugation, the microneedle negative mold was placed in a desiccator and dried at room temperature for 48 hours. The mold was carefully removed to obtain the whitening microneedle (HASL-GD) containing glycyrrhizin eutectic hydrogel. The microneedle was stored in a desiccator.

[0137] The morphology of the whitening microneedles in the glycyrrhizin inclusion complex eutectic hydrogel was observed by SEM, such as... Figure 10 As shown, the microneedle array is well-organized, forming a complete four-sided pyramid shape with sharp tips and uniform length.

[0138] Whitening microneedles containing glycyrrhizin as a matrix were prepared by replacing the combination of HA-Ser and HA-Lys with the same concentration of HA, HA-Ser, and HA-Lys, respectively, and were denoted as HA-GD, HAS-GD, and HAL-GD.

[0139] II. Determination of drug content in microneedles and drug utilization rate at the needle tip

[0140] Three glycyrrhizin inclusion complex eutectic hydrogel microneedles were taken and vortexed to dissolve in 3 mL of 10% Tween 80-PBS solution. After dilution by a certain factor, the GLA content in the entire microneedle was determined by HPLC. Then, the needle tip was carefully scraped off with a scalpel, and the remaining backing (substrate) was dissolved in 3 mL of 10% Tween 80-PBS solution. The solution was diluted and brought to volume using the same method, and the GLA content was measured. This process was repeated three times, and the average value was taken. The drug utilization rate of the needle tip / % = (Drug content of the entire microneedle - Drug content of the backing) / Drug content of the entire microneedle × 100%.

[0141] Test results: The drug loading of HASL-GD was 52.781±3.894 μg, and the utilization rate was approximately 89.78%±1.23%.

[0142] III. Determination of the mechanical strength of microneedles

[0143] The axial force mechanical properties of microneedles are the most commonly used experiment to evaluate their mechanical performance. Four types of microneedles—HASL-GD, HA-GD, HAS-GD, and HAL-GD—were prepared. The breaking force of the microneedles, i.e., the maximum force the microneedles can withstand under external force, was measured as an indicator of their mechanical strength. The microneedles were placed on the stage of a texture analyzer with the tip pointing upwards. The probe was gradually lowered at a speed of 0.1 mm / s. When the probe touched the tip of the microneedle, the mechanical changes during the stress period were recorded until the microneedle broke. The data were then plotted as a force-displacement curve.

[0144] The four types of microneedles prepared in this embodiment—HASL-GD, HA-GD, HAS-GD, and HAL-GD—were tested by pressing the microneedles with the sensor probe of a texture analyzer. The results showed that the modified HASL-GD (eutectic hydrogel microneedles) exhibited significantly enhanced mechanical properties compared to ordinary HA-GD microneedles. Furthermore, the HASL-GD prepared by combining HA-Ser and HA-Lys also showed significantly enhanced mechanical properties compared to HAS-GD or HAL-GD prepared by HA-Ser or HA-Lys alone (e.g., ...). Figure 11 ).

[0145] Example 5: In vitro transdermal experiment of glycyrrhizin inclusion complex eutectic hydrogel whitening microneedles.

[0146] The in vitro transdermal experiment used a modified Franz diffusion cell system, and set up a control group of HP-β-CD-GLA inclusion complex microneedles (prepared according to the method of Example 4 with HA-Ser and HA-Lys as microneedle matrices) and GLA-PBS (containing 0.5% ethanol) suspension for comparison.

[0147] Cumulative transdermal dose: Before the experiment, an appropriate amount of GLA was accurately weighed and dissolved in ethanol, then in phosphate buffered saline (PBS, pH 7.4). The solution was stirred for 12 h in the dark to prepare a drug suspension, ensuring that the dosage for each group was 380 μg GLA. The hairless abdominal skin of mice was fixed in a diffusion cell, with the stratum corneum facing the donor cell. For the microneedle group, the microneedle array was fixed with surgical tape, and a vertical pressure of 40 N was applied for 1 min to ensure stratum corneum penetration. For the suspension group, an equal volume of drug PBS solution was directly added to the donor cell. The receiving cell was filled with 10% Tween 80-PBS solution as the receiving medium. During the experiment, a constant temperature water bath of 37±0.5℃ was maintained, and magnetic stirring was performed at 300 rpm. 1 mL samples were taken at time points of 0.5, 1, 2, 4, 6, and 8 h, and isothermal blank medium was immediately added to maintain a constant volume in the receiving cell. After 1 h, all drug delivery systems were removed to simulate the actual transdermal process. The samples were filtered through a 0.45 μm microporous membrane and then quantitatively analyzed by high-performance liquid chromatography (HPLC). The cumulative transdermal absorption rate (Q, μg / cm²) was calculated using the formula Q = Cn×V + ΣCi×Vi, where Cn is the concentration of the nth sample, V is the volume of the receiving cell, Ci is the concentration of the i-th sample, and Vi is the sampling volume. A linear regression was performed on the cumulative transdermal absorption rate against time, and the slope of the fitted curve was the steady-state permeation rate (J, μg·cm²). -2 ·h -1 The x-intercept represents the osmosis lag (Tlag, h). All experiments were independently repeated three times, and data are presented as mean ± standard deviation (mean ± SD) to ensure the reliability of the results.

[0148] like Figure 12 As shown, the cumulative permeation rate (Q, μg·cm⁻¹) of HP-β-CD-GLA inclusion complex microneedles during transdermal treatment from 0.5 to 8 h was [data missing]. -2 The HP-β-CD-GLA inclusion complex showed significantly higher water solubility than GLA, allowing it to dissolve more quickly in the skin and diffuse into the receiving solution.

[0149] Two hours later, the transdermal rate (J) of the GLA / HP-β-CD inclusion complex microneedles was higher than that of the GLA PBS suspension, and it was able to be released continuously at a relatively stable rate, indicating that the inclusion complex microneedles have an advantage in prolonging the duration of drug action.

[0150] Skin retention: The skin drug retention behavior of HP-β-CD-GLA microneedles (prepared according to the method in Example 4 using HA-Ser and HA-Lys as microneedle matrices) and ordinary GLA-PBS (containing 0.5% ethanol) suspension was evaluated using the Franz diffusion cell method. Ex vivo rat abdominal skin was taken and laid flat on a dissecting plate with the stratum corneum facing upwards. HP-β-CD-GLA microneedles were placed vertically on the skin surface and acupressure was applied for 2 minutes. Subsequently, skin from the drug delivery area was cut to fit the size of the diffusion cell and immediately fixed between the supply and receiving cells using a horseshoe clamp. Another experimental group received an equal dose of GLA-PBS suspension as a control, which was evenly spread on the same skin area and fixed using the same method. 5 mL of PBS buffer (pH = 7) containing 10% Tween 80 was added to the receiving cell as the dissolution medium. The water bath temperature was maintained at 37 °C, and the stirring speed was set to 100 rpm. The device was placed in a transdermal diffusion apparatus, and timing was started. Skin samples were collected at 1, 2, and 4 hours. The microneedle base was gently peeled off, and the skin surface was wiped three times with a cotton swab moistened with physiological saline to remove residual preparation. The remaining moisture was then absorbed with a dry cotton swab. The skin was minced and placed in a tissue homogenizer. 1 mL of chromatographic grade methanol was added, and the mixture was ground until no large pieces of tissue were visible and a flocculent suspension was formed, ensuring sufficient drug transfer to the methanol. The mixture was centrifuged at 4 ℃ and 4000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm microporous membrane, and the glycyrrhizin content was determined according to the established chromatographic method. A bar chart of drug retention on the skin at different time points was then plotted.

[0151] The results are as follows Figure 13 As shown, the skin retention of the HP-β-CD-GLA microneedle group was significantly higher than that of the ordinary suspension group. This difference is mainly due to the unique advantages of the microneedle delivery system: 1) the microneedle array can physically penetrate the stratum corneum barrier to achieve targeted delivery to the dermis; 2) the soluble microneedle matrix can maintain sustained drug release and prolong the duration of action; 3) the closed-loop drug delivery of microneedles avoids the loss caused by transdermal penetration of traditional formulations. Experimental data confirm that the microneedles of this invention can significantly improve the transdermal delivery efficiency of glycyrrhizin, providing an effective solution to the problem of low transdermal absorption of this component.

[0152] Example 6 In vitro pharmacodynamic experiment

[0153] 1. In vitro antioxidant experiment

[0154] The ABTS radical scavenging capacity was determined using the 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) method. This method is a widely used standard indirect detection method suitable for assessing the antioxidant capacity of both hydrophilic and lipophilic substances. Its principle is based on the fact that ABTS generates stable blue-green cationic radicals (ABTS·) in the presence of oxidants (such as potassium persulfate). When antioxidants are present in the system, they can scavenge ABTS·, causing the reaction system to decolorize and exhibit a decrease in absorbance at 405 nm. Within a certain concentration range, the decrease in absorbance is proportional to the amount of free radicals scavenged, thus quantitatively reflecting the ability of the sample to scavenge ABTS· radicals. The specific experimental steps are as follows: First, blank microneedles (denoted as HA, HAS, HAL, and HASL in this example) with HA, HA-Ser, HA-Lys, and combinations of HA-Ser and HA-Lys as the matrix, and drug-containing microneedles loaded with glycyrrhizin-HP-β-CD (denoted as SLGD in this example) with a combination of HA-Ser and HA-Lys as the matrix were prepared according to the method in Example 4. The matrix content of each group of microneedles was equal. Then, the tips of each group of microneedles were scraped off with a knife and dissolved in 1 ml of water to obtain microneedle extract as the test sample. Subsequently, a 96-well microplate was prepared, and blank wells (using water instead of the sample), control wells (containing ABTS working solution but not the sample), and sample wells were set up. Each component was added to the corresponding well strictly according to the instructions of the ABTS free radical scavenging ability test kit. After the sample was added, the contents of each well were thoroughly mixed and allowed to stand at room temperature in the dark for 6 minutes. Finally, the absorbance of each well was measured at a wavelength of 405 nm using a microplate reader (denoted as A). 空白 A 对照 and A 测定 )

[0155] The ABTS* free radical scavenging rate (%) is calculated using the following formula:

[0156] ABTS*·Free radical scavenging rate (%) = [A 空白 -(A 测定 -A 对照 )] / A 空白 *100%

[0157] like Figure 14As shown, the ABTS⁺· free radical scavenging abilities of serine-modified hyaluronic acid (HAS) and lysine-modified hyaluronic acid (HAL) are both higher than those of unmodified hyaluronic acid (HA). The composite hydrogel (HASL) formed by the two exhibits a more significant scavenging ability. The glycyrrhizin inclusion complex eutectic hydrogel whitening microneedles (SLGD) prepared with it as the matrix can significantly improve the free radical scavenging ability of glycyrrhizin.

[0158] 2. Determination of ROS scavenging and related enzyme expression

[0159] Reactive oxygen species (ROS) are natural byproducts of normal intracellular oxygen metabolism, primarily originating from the mitochondrial respiratory chain. Under appropriate conditions, ROS contribute to cellular immunity, repair, and growth. However, when ROS levels are excessively high, exceeding the cell's antioxidant defense capacity, they can lead to structural or conformational changes in DNA, lipids, and proteins, resulting in cell damage and death, causing cellular oxidative stress. The fluorescent probe DCFH-DA, after entering the cell, is hydrolyzed by esterases to generate 2',7'-dichlorodihydrofluorescein, which is then rapidly oxidized by ROS to generate the strongly fluorescent product 2,7-chlorofluorescein. Detection of 2',7'-dichlorofluorescein reveals the intracellular ROS level. This embodiment uses laser confocal microscopy (CLSM) to detect intracellular reactive oxygen species (ROS) levels. The specific steps are as follows: HacaT cells are subjected to a 4×10⁻⁶... 4 Cells were seeded at a density of 80%–90% in 12-well laser confocal microscopy plates. Once cell confluence reached 80%–90%, the following experimental groups were established: a blank control group (Control), a model group (UVA irradiation), a HA group, a HASL group, and a SLGD group (the latter three groups were treated with the drug after UVA irradiation; the preparation of the test samples was the same as in the in vitro antioxidant experiment described in section 1). Cells in each group were co-incubated with the corresponding drug at 37°C and 5% CO2 for 12 hours. The drug-containing medium was then discarded, and the DCFH-DA fluorescent probe diluted to 100 μM with DMEM was added. The cells were incubated in the dark for 30 minutes. Subsequently, the cells were gently washed three times with PBS, and 1 mL of PBS was added to each well. Immediately afterward, the cells were observed and images were acquired under a laser confocal microscope.

[0160] The results are as follows Figure 15 As shown, the ROS level in HaCaT cells significantly increased after UV irradiation, successfully simulating cellular oxidative stress. After incubation with different drug groups, ROS levels decreased to varying degrees. The modified hyaluronic acid microneedle extract group (HASL) showed better ROS scavenging effect than the HA microneedle extract group alone. Furthermore, the SLGD (microneedle extract containing glycyrrhizin inclusion complex) group demonstrated the best ROS scavenging and oxidative stress resistance effect compared to the Model group.

[0161] To test the antioxidant properties of the HSAL (pure microneedle matrix) and SLGD (microneedle matrix containing glycyrrhizin inclusion complex), a human immortalized keratinocyte (HaCaT) model was used. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured to evaluate its antioxidant performance. The specific steps were as follows: After routine culture, HaCaT cells were randomly divided into a blank control group (Control), a Model group (UV irradiation), a HA group (5 mg / mL), a HSAL (5 mg / mL) treatment group, a positive control group (Arbutin group, 10 μM), a GLA group (10 μM), and an SLGD (containing 10 μM GLA) treatment group. Except for the blank control group, all other groups were irradiated with UV light for 5 minutes, followed by the addition of the corresponding drug intervention, and incubated for another 24 hours. After the intervention, cells were collected, and total protein was extracted using lysis buffer. Superoxide dismutase (SOD) activity was detected using the WST-8 method, and MDA content was determined using the thiobarbituric acid (TBA) method. The procedures were strictly performed according to the kit instructions. Experiments were independently repeated three times. Data are expressed as mean ± standard deviation, and statistical comparisons were performed using one-way ANOVA.

[0162] result( Figure 16 The results showed that SOD activity was significantly increased in the UVB radiation group (p<0.05), accompanied by an increase in MDA content. This phenomenon suggests that UVB-induced oxidative stress leads to the accumulation of intracellular superoxide free radicals, triggering lipid peroxidation and causing cellular oxidative damage. In contrast, cells treated with HASL and SLGD groups exhibited significant antioxidant effects: both groups effectively upregulated SOD activity and reduced MDA levels (p<0.001), indicating that they can alleviate oxidative damage by scavenging excess ROS. Furthermore, the regulatory effects of SLGD and HASL groups on SOD and MDA were significantly better than those of the HA group, indicating that the modified HASL group has a stronger free radical scavenging capacity and membrane lipid protection effect than the HA group. These results collectively demonstrate that SLGD can significantly improve the antioxidant defense system of HaCaT cells through multi-target regulation.

[0163] 3. Anti-inflammatory experiment

[0164] In a photoaging model, the expression levels of inflammation-related cytokines were detected using an ELISA kit. The specific experimental steps were as follows: HaCaT cells were routinely cultured and then randomly divided into a blank control group (Control), a model group (UV irradiation), a HA group (5 mg / mL), a HSAL group (5 mg / mL), a positive control group (arbutin, 10 μM), a GLA group (10 μM), and an SLGD group (containing 10 μM glycyrrhizin). Except for the blank control group, all other groups were irradiated with UV light for 5 minutes, followed by intervention with the corresponding drug, and incubated for 24 hours. After intervention, the cell supernatant was collected, and the ELISA kit was strictly followed according to the instructions. The experiment was independently repeated three times, and the data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. It was found that HASL and SLGD (the preparation of the test samples was the same as in the in vitro antioxidant experiment in 1) could significantly inhibit the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6, and the anti-inflammatory effect of SLGD was better than that of the positive control drugs arbutin and glycyrrhizin alone. Figure 17 Further studies have demonstrated that the modified hyaluronic acid has better biological functions, and the glycyrrhizin inclusion complex eutectic hydrogel whitening microneedles (SLGD) prepared using it as a matrix can significantly improve the anti-inflammatory effect of glycyrrhizin.

[0165] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A glycyrrhizin inclusion complex hydrogel whitening microneedle, comprising a needle tip and a base, characterized in that, The needle tip was prepared from an aqueous solution of glycyrrhizin-HP-β-CD inclusion complex and hyaluronic acid derivative; The glycyrrhizin-HP-β-CD inclusion complex was obtained by encapsulating glycyrrhizin with hydroxypropyl-β-cyclodextrin; The hyaluronic acid derivative is composed of L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid; The L-serine-grafted hyaluronic acid is obtained by an amidation reaction of hyaluronic acid and L-serine, and the L-lysine-grafted hyaluronic acid is obtained by an amidation reaction of hyaluronic acid and L-lysine. The molecular weight of the hyaluronic acid is 300,000 to 1,000,000 The mass ratio of hyaluronic acid to L-serine is 1.5-2:1; The mass ratio of hyaluronic acid to L-lysine is 1-1.5:

1.

2. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 1, characterized in that, The mass ratio of the glycyrrhizin-HP-β-CD inclusion complex to the hyaluronic acid derivative is 1:1-3; And / or, the mass fraction of the hyaluronic acid derivative in the aqueous solution of the glycyrrhizin-HP-β-CD inclusion complex and the hyaluronic acid derivative is 15%-25%.

3. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 2, characterized in that, The mass ratio of the glycyrrhizin-HP-β-CD inclusion complex to the hyaluronic acid derivative is 1:1.5-2.5; And / or, the mass fraction of the hyaluronic acid derivative in the aqueous solution of the glycyrrhizin-HP-β-CD inclusion complex and the hyaluronic acid derivative is 18%-22%.

4. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 3, characterized in that, The mass ratio of the glycyrrhizin-HP-β-CD inclusion complex to the hyaluronic acid derivative is 1:1.8-2.

2.

5. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 1, characterized in that, The mass ratio of L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid in the hyaluronic acid derivative is 1:0.5-1.

5.

6. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 5, characterized in that, The mass ratio of L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid in the hyaluronic acid derivative is 1:0.8-1.

2.

7. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 1, characterized in that, The molecular weight of the hyaluronic acid is 400,000 to 800,000.

8. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 1, characterized in that, The preparation method of the L-serine-grafted hyaluronic acid or L-lysine-grafted hyaluronic acid includes the following steps: The hyaluronic acid is dissolved in a buffer solution, an activator is added to activate the carboxyl group of the hyaluronic acid, and then L-serine or L-lysine is added. The reaction is carried out to obtain L-serine-grafted hyaluronic acid or L-lysine-grafted hyaluronic acid.

9. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 8, characterized in that, The pH of the buffer solution is 4.5-5.

5.

10. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 8, characterized in that, The buffer solution is a MES buffer solution.

11. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 8, characterized in that, The activator is carbodiimide and N-hydroxysuccinimide.

12. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 11, characterized in that, The mass ratio of the carbodiimide to N-hydroxysuccinimide is 1.5-2:

1.

13. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 8, characterized in that, The amount of the activator is 0.9-1.2 times the weight of the hyaluronic acid.

14. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 8, characterized in that, The activation temperature is 40℃-50℃, and the time is 10min-20min.

15. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 8, characterized in that, The reaction is carried out at a temperature of 40℃-50℃ for a time of 20h-28h.

16. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 1, characterized in that, The glycyrrhizin-HP-β-CD inclusion complex was prepared by glycyrrhizin and hydroxypropyl-β-cyclodextrin in a molar ratio of 1:1.2-1.

8.

17. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 16, characterized in that, The preparation method of the glycyrrhizin-HP-β-CD inclusion complex includes the following steps: The glycyrrhizin and hydroxypropyl-β-cyclodextrin were prepared into ethanolic solutions of glycyrrhizin and aqueous solutions of hydroxypropyl-β-cyclodextrin with concentrations of 0.3 mol / L to 0.32 mol / L, respectively. The ethanolic solution of glycyrrhizin was slowly titrated into the aqueous solution of hydroxypropyl-β-cyclodextrin at a constant temperature of 35℃ to 45℃ until a transient white precipitate appeared, at which point the titration was stopped. The reaction system was stirred continuously for 20 h to 28 h, the ethanol was removed, the unencapsulated drug was removed by filtration, and the mixture was lyophilized to obtain the glycyrrhizin-HP-β-CD inclusion complex.

18. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to any one of claims 1-17, characterized in that, The substrate is prepared from an aqueous solution of oligomeric hyaluronic acid.

19. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 18, characterized in that, The oligomeric hyaluronic acid has a molecular weight of less than 20 kDa.

20. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 19, characterized in that, The oligomeric hyaluronic acid has a molecular weight of less than 10 kDa.

21. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 18, characterized in that, The mass concentration of the oligomeric hyaluronic acid aqueous solution is 15%-25%.

22. The glycyrrhizin inclusion complex hydrogel whitening microneedle according to claim 21, characterized in that, The mass concentration of the oligomeric hyaluronic acid aqueous solution is 18%-22%.

23. A method for preparing a whitening microneedle of glycyrrhizin inclusion complex hydrogel according to any one of claims 1-22, characterized in that, Includes the following steps: (1) Dissolve the glycyrrhizin-HP-β-CD inclusion complex in water to prepare a homogeneous aqueous solution, then add the L-serine-grafted hyaluronic acid and L-lysine-grafted hyaluronic acid, mix evenly to obtain a needle tip solution; (2) Add the needle tip solution to the microneedle negative mold, centrifuge to fill the microchannels of the negative mold with the needle tip solution, scrape off the needle tip solution on the surface of the microneedle negative mold, centrifuge again to fully compress the needle tip solution into the microchannels of the mold; (3) Dry the microneedle negative mold containing the needle tip solution obtained in step (2); (4) Take the base solution and spread it evenly on the microneedle negative mold after drying in step (3), centrifuge, dry, and take out the microneedle negative mold at certain intervals during the drying process, add base solution and centrifuge, so that the base solution completely covers the micropores; (5) After adding the base solution for the last time and centrifuging, the microneedle negative mold is dried and demolded to obtain the glycyrrhizin inclusion complex hydrogel whitening microneedle.

24. The method for preparing the glycyrrhizin inclusion complex hydrogel whitening microneedles according to claim 23, characterized in that, The centrifugation conditions described in step (2) include: 3500 rpm-4500 rpm, 4℃-10℃, 25min-35min.

25. The method for preparing the glycyrrhizin inclusion complex hydrogel whitening microneedles according to claim 23, characterized in that, Repeat step (2) 2-4 times.

26. The method for preparing the glycyrrhizin inclusion complex hydrogel whitening microneedles according to claim 23, characterized in that, The drying temperature in step (3) is 15℃-40℃, and the time is 20 hours-28 hours.

27. The method for preparing the glycyrrhizin inclusion complex hydrogel whitening microneedles according to claim 23, characterized in that, The centrifugation conditions in step (4) include: 3500 rpm-4500 rpm, 4℃-10℃, 8min-15min.

28. The method for preparing the glycyrrhizin inclusion complex hydrogel whitening microneedles according to claim 23, characterized in that, In step (4), "every certain time" means every 3-5 hours, adding the base solution and centrifuging 2-5 times.

29. The method for preparing the glycyrrhizin inclusion complex hydrogel whitening microneedles according to claim 23, characterized in that, The drying temperature in step (4) is 15℃-40℃.

30. The method for preparing the glycyrrhizin inclusion complex hydrogel whitening microneedles according to claim 23, characterized in that, The drying temperature in step (5) is 15℃-40℃, and the time is 40 hours-56 hours.

31. The use of the glycyrrhizin inclusion complex hydrogel whitening microneedles as described in any one of claims 1-30 in the preparation of whitening, antioxidant and / or anti-inflammatory products.

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