A composition for treating alopecia, microneedles and a preparation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a composition for treating hair loss, microneedles, their preparation method, and applications. The composition for treating hair loss comprises arginine and pentacyclic triterpenoids; the composition is a co-amorphous substance. The arginine and pentacyclic triterpenoids exhibit a synergistic effect, significantly improving the therapeutic effect on hair loss compared to using the active ingredients alone or as a suspension formed by direct mixing. The co-amorphous system formed by arginine and pentacyclic triterpenoids can upregulate the Wnt / β-catenin pathway, improve the microenvironment of oxidative stress and insufficient angiogenesis around hair follicles, providing favorable conditions for hair follicle growth and effectively preventing and treating hair loss. Furthermore, further processing the co-amorphous system into microneedles can enhance the therapeutic effect. Simultaneously, the separable design of the microneedles allows the drug to be effectively retained in the skin and continuously released, while reducing the frequency of drug administration.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and specifically relates to a composition for treating hair loss, microneedles, their preparation method and application. Background Technology

[0002] Hair loss is a common disease in contemporary society, with complex and diverse causes encompassing multiple aspects such as physiological aging, disease effects, and social stress. In recent years, the incidence of hair loss has been rising year by year and showing a trend towards affecting younger people, seriously impacting the physical and mental health of patients.

[0003] Currently, treatment options for hair loss are limited, primarily including medication, physical therapy (such as low-energy laser and scalp microneedling), and surgical treatment (such as hair transplantation). However, these treatments all have limitations in terms of cost, effectiveness, and safety. For example, the U.S. Food and Drug Administration (FDA) has only approved topical minoxidil and oral finasteride for treating hair loss. While these medications can alleviate symptoms to some extent, their side effects are significant. For instance, the ethanol and propylene glycol in minoxidil formulations can cause scalp dryness and irritation, while the oral form of finasteride may cause adverse reactions such as sexual dysfunction. These side effects not only affect the patient's treatment experience but may also threaten their overall health. Therefore, there is an urgent need for safer and more effective treatments to alleviate the distress caused by hair loss.

[0004] Therefore, it is of great significance to provide a composition that effectively promotes hair growth and is green and safe for treating hair loss. Summary of the Invention

[0005] The present invention aims to solve one or more technical problems existing in the prior art, and at least provide a beneficial option or create conditions. Specifically, the present invention provides a composition for treating hair loss, which has a good effect on promoting hair growth, can significantly improve the treatment effect of hair loss, and is green and safe.

[0006] The inventive concept of this invention: The composition for treating hair loss of this invention comprises arginine and pentacyclic triterpenoids; the composition is a co-amorphous compound. The arginine and pentacyclic triterpenoids of this invention have a synergistic effect, significantly improving the therapeutic effect on hair loss compared to using the active ingredient alone, and compared to a suspension formed by the mixture of arginine and pentacyclic triterpenoids. Furthermore, the co-amorphous system formed by arginine and pentacyclic triterpenoids can upregulate the Wnt / β-catenin pathway, improve the microenvironment of oxidative stress and insufficient angiogenesis around the hair follicle, providing favorable conditions for hair follicle growth and effectively preventing and treating hair loss.

[0007] Therefore, a first aspect of the present invention provides a composition for treating hair loss.

[0008] Specifically, the composition for treating hair loss includes arginine and pentacyclic triterpenoids; the composition is a co-amorphous compound.

[0009] Specifically, compared to suspensions of arginine and pentacyclic triterpenoids, the amorphous composition can enhance the transdermal delivery efficiency of the active ingredients arginine and pentacyclic triterpenoids, promote the proliferation of human dermal papilla cells, reverse the effects of dihydrotestosterone on the expression of various factors in human dermal follicular papilla cells, resist oxidative stress, and promote the migration of human umbilical vein endothelial cells, thus having a better therapeutic effect on hair loss.

[0010] Co-amorphous compounds are unidirectional amorphous systems composed of two or more small molecule components. These components are connected by non-covalent bonds such as hydrogen bonds, ionic bonds, and π-π stacking, or may not have any interaction forces. Using a co-amorphous form can significantly improve the solubility and dissolution rate of poorly soluble drugs, and also enhance the physical stability of amorphous drugs. Administering drugs in a co-amorphous form after combining two small molecules with active ingredients can fully leverage the advantages of using arginine and pentacyclic triterpenoids in combination for treating hair loss, significantly improving the treatment effect compared to using the active ingredient alone. Furthermore, co-amorphous systems have the unique property of forming gels in water, which can improve the hydrophobicity of pentacyclic triterpenoids and facilitate the processing into other formulations.

[0011] Preferably, the pentacyclic triterpenoid compound includes at least one of oleanolic acid, glycyrrhetinic acid, ursolic acid, asiatic acid, betulinic acid, and hawthorn acid.

[0012] Preferably, the molar ratio of arginine to pentacyclic triterpenoid is (0.9-4.5):1; more preferably, the molar ratio of arginine to pentacyclic triterpenoid is (1-4):1.

[0013] A second aspect of the present invention provides a method for preparing the composition for treating hair loss described in the first aspect of the present invention.

[0014] Specifically, the preparation method of the hair loss treatment composition includes the following steps:

[0015] The raw material components are mixed, ultrasonically treated, and dried to obtain the composition.

[0016] Preferably, the method for preparing the hair loss treatment composition includes the following steps:

[0017] (1) Arginine and water are mixed to obtain an arginine solution; a pentacyclic triterpenoid compound, an alcohol and / or an ether are mixed to obtain a dispersion of the pentacyclic triterpenoid compound;

[0018] (2) The arginine solution obtained in step (1) and the dispersion of the pentacyclic triterpenoid compound are mixed, ultrasonically treated, and dried to obtain the composition.

[0019] Preferably, in step (1), the alcohol includes at least one of anhydrous ethanol and methanol; more preferably, the alcohol includes anhydrous ethanol.

[0020] Preferably, in step (1), the ether includes diethyl ether.

[0021] Preferably, in step (2), the ultrasonic treatment time is 20-40 min; more preferably, the ultrasonic treatment time is 28-33 min; and even more preferably, the ultrasonic treatment time is 30 min.

[0022] Preferably, in step (2), the drying process includes rotary evaporation and vacuum drying in sequence.

[0023] Preferably, the rotary evaporation temperature is 35-50℃ and the rotary evaporation time is 9-22 min; more preferably, the rotary evaporation temperature is 40-50℃ and the rotary evaporation time is 10-20 min.

[0024] Preferably, the vacuum drying temperature is 30-50℃ and the vacuum drying time is 40-55h; more preferably, the vacuum drying temperature is 35-45℃ and the vacuum drying time is 45-52h; even more preferably, the vacuum drying temperature is 40℃ and the vacuum drying time is 48h.

[0025] Specifically, the composition obtained by the above preparation method is an amorphous substance. Compared with directly mixing arginine and pentacyclic triterpenoids to prepare a suspension, it can significantly improve the transdermal delivery efficiency of the active ingredients arginine and pentacyclic triterpenoids, thereby improving the effect of treating hair loss.

[0026] A third aspect of the present invention provides a hydrogel.

[0027] Specifically, the hydrogel comprises the hair loss treatment composition described in the first aspect of the present invention.

[0028] Preferably, the method for preparing the hydrogel includes the following steps:

[0029] The hair loss treatment composition and solvent are mixed and ultrasonically treated to obtain the hydrogel.

[0030] Specifically, ultrasonic treatment can enable compositions with amorphous systems to self-assemble into hydrogels.

[0031] A fourth aspect of the present invention provides a microneedle.

[0032] Specifically, the microneedle sequentially comprises a backing layer, a separation layer, and a needle tip; the needle tip is composed of the hydrogel described in the third aspect of the present invention.

[0033] Preferably, the mass ratio of the backing layer, the separating layer, and the needle tip is (1.5-4.5):(0.8-1.2):1; more preferably, the mass ratio of the backing layer, the separating layer, and the needle tip is (1.8-2.5):(0.9-1.1):1; even more preferably, the mass ratio of the backing layer, the separating layer, and the needle tip is 2:1:1.

[0034] Preferably, the backing layer comprises a solvent and a biodegradable polymer material.

[0035] Preferably, the biodegradable polymer material in the backing layer includes at least one of hyaluronic acid and dextran; more preferably, the biodegradable polymer material in the backing layer includes hyaluronic acid and dextran.

[0036] Preferably, in the backing layer, the hyaluronic acid comprises 200-400kDa hyaluronic acid and 37-56kDa hyaluronic acid.

[0037] Preferably, the separation layer comprises a solvent, a biodegradable polymer material, and sucrose.

[0038] Preferably, in the separation layer, the biodegradable polymer material includes at least one of hyaluronic acid, sodium carboxymethyl cellulose, dextran, and polyvinyl alcohol; more preferably, in the separation layer, the biodegradable polymer material includes hyaluronic acid with a content of 3-10 kDa.

[0039] Preferably, the needle tip also comprises a biodegradable polymer material.

[0040] Preferably, the biodegradable polymer material in the needle tip includes dextran.

[0041] The fifth aspect of the present invention provides a method for preparing the microneedles described in the fourth aspect of the present invention.

[0042] Specifically, the preparation method of the microneedles includes the following steps:

[0043] The needle tip, the separation layer, and the backing layer are sequentially formed in a mold to obtain the microneedle.

[0044] The sixth aspect of the present invention provides the use of the composition for treating hair loss described in the first aspect of the present invention, the hydrogel described in the third aspect of the present invention, or the microneedles described in the fourth aspect of the present invention in the preparation of a medicament for treating hair loss.

[0045] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0046] (1) The composition for treating hair loss of the present invention comprises arginine and pentacyclic triterpenoids; the composition is a co-amorphous compound. The arginine and pentacyclic triterpenoids of the present invention have a synergistic effect, significantly improving the therapeutic effect on hair loss compared to the use of the active ingredient alone, and compared to a suspension formed by the mixture of arginine and pentacyclic triterpenoids. Furthermore, the co-amorphous system formed by arginine and pentacyclic triterpenoids can upregulate the Wnt / β-catenin pathway, improve the microenvironment of oxidative stress and insufficient angiogenesis around the hair follicle, providing favorable conditions for hair follicle growth and effectively preventing and treating hair loss.

[0047] (2) The composition of this invention is a co-amorphous material, which has the property of self-assembling into a supramolecular hydrogel in water, providing convenience for further formulation design (e.g., the gel form facilitates the fabrication of microneedles, and can be further designed into thermosensitive gels, pH-responsive gels, injectable gels, etc. by adding other materials). Simultaneously, this invention further loads the hydrogel into separable microneedles. The microneedles are designed to be separable, enabling rapid separation of the needle tip and backing layer, allowing the drug in the needle tip to remain in the skin for slow release. This not only improves drug delivery efficiency but also forms a drug reservoir under the skin, achieving the goal of long-term treatment of hair loss. This invention is the first to combine co-amorphous drugs with microneedles, expanding the formulation forms and administration methods of co-amorphous drugs, and providing new ideas and directions for their future development and application.

[0048] (3) The composition of the present invention does not contain irritating ingredients such as ethanol and propylene glycol, making it greener and safer; at the same time, it can effectively reduce the frequency of administration and reduce the pain caused by frequent administration; in addition, the preparation process of the present invention is simple and convenient for large-scale production and application. Attached Figure Description

[0049] Figure 1 This is an image of the hydrogel prepared in Example 4 of the present invention.

[0050] Figure 2 This is an appearance diagram of the microneedles prepared in Application Example 1 of the present invention;

[0051] Figure 3 X-ray diffraction pattern of the hair loss treatment composition prepared in Example 1 of the present invention;

[0052] Figure 4This is a transmission electron microscope image of the hydrogel prepared in Example 4 of the present invention;

[0053] Figure 5 This is a rheological curve of the hydrogel prepared in Example 4 of the present invention;

[0054] Figure 6 The mechanical properties of the microneedles prepared in Application Example 1 of this invention are shown in the diagram.

[0055] Figure 7 This is a graph showing the porosity of the sealing film of the microneedles prepared in Application Example 1 of this invention;

[0056] Figure 8 This is a diagram showing the microneedle separation results of the microneedles prepared in Application Example 1 of the present invention;

[0057] Figure 9 The in vitro drug release performance of oleanolic acid in the microneedles prepared in Application Example 1 of this invention is shown in the figure.

[0058] Figure 10 The diagram shows the effect of the composition of Example 1 of the present invention, arginine, and oleanolic acid on the proliferation of human dermal hair follicle papillary cells.

[0059] Figure 11 The relative expression level of CTNNBIP1 mRNA in human dermal hair follicle papillary cells is shown in the figure.

[0060] Figure 12 A graph showing the relative expression levels of DKK1 mRNA in human dermal hair follicle papillary cells;

[0061] Figure 13 The relative expression level of TGFB1 mRNA in human dermal hair follicle papillary cells is shown in the figure.

[0062] Figure 14 A graph showing the relative expression levels of IGF1 mRNA in human dermal hair follicle papillary cells;

[0063] Figure 15 Figure showing the survival rate of human dermal hair follicle papillary cells in each treatment group under hydrogen peroxide conditions;

[0064] Figure 16 The image shows the fluorescence detection results of reactive oxygen species (ROS) produced by human dermal follicular papillary cells in each treatment group under hydrogen peroxide conditions.

[0065] Figure 17 The graph shows the effect of each treatment group on promoting the migration of human umbilical vein endothelial cells.

[0066] Figure 18 A graph showing the statistical results of the migration area of ​​human umbilical vein endothelial cells in each treatment group;

[0067] Figure 19 Figure 1 shows the hair growth on the backs of mice in each group during the drug administration period;

[0068] Figure 20 This is a statistical graph showing the hair regeneration coverage rate of mice in each group after drug administration.

[0069] Figure 21 This is a statistical graph showing the length of regenerated hair in mice in each group after drug administration. Detailed Implementation

[0070] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0071] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0072] Example 1

[0073] A composition for treating hair loss includes 0.27g of arginine and 0.36g of oleanolic acid.

[0074] A method for preparing a composition for treating hair loss includes the following steps:

[0075] (1) Dissolve 0.27g of arginine in 2mL of water to obtain an arginine solution; disperse 0.36g of oleanolic acid in 40mL of anhydrous ethanol by ultrasonication to obtain a dispersion of oleanolic acid.

[0076] (2) Slowly add arginine solution to oleanolic acid dispersion, sonicate at room temperature for 30 minutes to obtain colorless and transparent solution; then evaporate the solution at 45°C to remove most of the water and ethanol, and then place the obtained product in vacuum drying at 40°C for 48 hours to obtain composition for treating hair loss.

[0077] Example 2

[0078] A composition for treating hair loss includes 0.27g of arginine and 0.74g of glycyrrhetinic acid.

[0079] A method for preparing a composition for treating hair loss includes the following steps:

[0080] (1) Dissolve 0.27g of arginine in 2mL of water to obtain an arginine solution; disperse 0.74g of glycyrrhetinic acid in 40mL of anhydrous ethanol by ultrasonication to obtain a glycyrrhetinic acid dispersion.

[0081] (2) Slowly add arginine solution to the dispersion of glycyrrhetinic acid, sonicate at room temperature for 30 minutes to obtain a colorless and transparent solution; then evaporate the solution at 45°C to remove most of the water and ethanol, and then place the obtained product under vacuum at 40°C for 48 hours to obtain a composition for treating hair loss.

[0082] Example 3

[0083] A composition for treating hair loss includes 0.54g of arginine and 0.36g of ursolic acid.

[0084] A method for preparing a composition for treating hair loss includes the following steps:

[0085] (1) Dissolve 0.54g of arginine in 2mL of water to obtain an arginine solution; disperse 0.36g of ursolic acid in 40mL of anhydrous ethanol by ultrasonication to obtain a ursolic acid dispersion.

[0086] (2) Slowly add arginine solution to ursolic acid dispersion, sonicate at room temperature for 30 minutes to obtain colorless and transparent solution; then evaporate the solution at 45°C to remove most of the water and ethanol, and then place the obtained product under vacuum at 40°C for 48 hours to obtain composition for treating hair loss.

[0087] Example 4

[0088] A hydrogel comprising 0.06 g of the hair loss treatment composition prepared in Example 1 and 1 g of water.

[0089] The preparation method of hydrogel includes the following steps:

[0090] The hair loss treatment composition prepared in Example 1 was added to water, stirred evenly, and then sonicated for 1 hour to allow it to self-assemble, thus obtaining a hydrogel.

[0091] The appearance of the hydrogel obtained in Example 4 is shown in the figure below. Figure 1 As shown, where, Figure 1 Figures (a) and (b) in the figure show the appearance of the hydrogel of Example 4 under different placement conditions.

[0092] Example 5

[0093] A hydrogel comprising 0.1 g of the hair loss treatment composition prepared in Example 2 and 1 g of water.

[0094] The preparation method of hydrogel includes the following steps:

[0095] The hair loss treatment composition prepared in Example 2 was added to water, stirred evenly, and then sonicated for 1 hour to allow it to self-assemble, thus obtaining a hydrogel.

[0096] Example 6

[0097] A hydrogel comprising 0.1g of the hair loss treatment composition prepared in Example 3 and 1g of water.

[0098] The preparation method of hydrogel includes the following steps:

[0099] The hair loss treatment composition prepared in Example 3 was added to water, stirred evenly, and then sonicated for 0.5 h to allow it to self-assemble and obtain a hydrogel.

[0100] Application Example 1

[0101] A separable microneedle comprises, in sequence, a backing layer, a separation layer, and a needle tip.

[0102] The backing layer consists of 0.1g of hyaluronic acid with a molecular weight of 200-400kDa, 0.1g of hyaluronic acid with a molecular weight of 37-56kDa, 0.1g of dextran, and 1mL of water.

[0103] The method for preparing the matrix of the backing layer includes the following steps:

[0104] Two molecular weight hyaluronic acid and dextran were added to water, stirred thoroughly, and placed in the refrigerator overnight to allow them to fully swell, thus obtaining the matrix of the backing layer. Before use, the air bubbles were removed by centrifugation.

[0105] The separation layer consists of 0.4g of hyaluronic acid with a molecular weight of 3-10kDa, 0.1g of sucrose, and 1mL of water.

[0106] The method for preparing the matrix of the separation layer includes the following steps:

[0107] Hyaluronic acid and sucrose are added to water and stirred thoroughly. The mixture is then placed in the refrigerator overnight to allow it to fully swell, resulting in the matrix of the separation layer. Before use, air bubbles are removed by centrifugation.

[0108] The composition of the needle tip (the composition of the needle tip matrix) includes 1.06 g of the hydrogel prepared in Example 4 and 0.12 g of dextran.

[0109] The method for preparing the matrix of the needle tip includes the following steps:

[0110] Add dextran to the hydrogel prepared in Example 4, stir evenly, and place in the refrigerator overnight to allow it to fully swell, thus obtaining the matrix of the needle tip. Remove air bubbles by centrifugation before use.

[0111] A method for preparing separable microneedles includes the following steps:

[0112] (1) Preparation of needle tip: Add the needle tip matrix to the polydimethylsiloxane (PDMS) mold, centrifuge at 3750 rpm at 4℃ for 2-4 times, 3 min each time, and change the direction of the mold to make the needle tip matrix fill the needle tip cavity of the mold; scrape off the excess needle tip matrix with a flat spatula, centrifuge at 3750 rpm at 25℃ for 30 min to make the needle tip matrix fully deposited and dry, and obtain the needle tip;

[0113] (2) Preparation of the separation layer: The matrix of the separation layer was added into the PDMS mold and centrifuged at 3750 rpm at 25°C for 30 min to allow the matrix of the separation layer to be fully deposited and dried to obtain the separation layer;

[0114] (3) Preparation of backing layer: Take the matrix of the backing layer and add it into the PDMS mold. Centrifuge twice at 3750 rpm at 4℃ for 3 min each time. At the same time, change the direction of the mold to make the matrix of the backing layer spread flat in the mold to obtain the backing layer. Then put the whole into the desiccator and dry it at 4℃ for 48 h. Demold to obtain separable microneedles.

[0115] The appearance of the microneedle is shown in the figure. Figure 2 As shown. Among them, Figure 2 Figure (a) in the image is a macroscopic view of the microneedles taken by a camera; Figure 2 Figure (b) shows a microneedle image taken under a microscope; Figure 2 Figure (c) in the image is a scanning electron microscope image of the microneedles; Figure 2 Figure (d) shows microneedle images taken under natural light after the tips and separation layers of the microneedles were labeled with different fluorescent dyes. Figure 2 Figure (e) in the image shows the tip of a microneedle under a fluorescence microscope; Figure 2 Figure (f) in the image shows the separation layer of the microneedles under a fluorescence microscope; Figure 2 Figure (g) shows an image of the combined tip of the microneedle and the separation layer under a fluorescence microscope.

[0116] Depend on Figure 2 It can be seen that the tip of the microneedle is a regular square pyramid + regular square prism shape, with a total tip length of 1200 μm, a base width of 300 μm, and a spacing of 600 μm between microneedles. When sodium fluorescein (emitting green fluorescence) and rhodamine B (emitting red fluorescence) were loaded onto the separation layer and tip of the microneedles, respectively, a distinct bilayer structure of the microneedles could be observed under both natural light and a fluorescence microscope.

[0117] Comparative Example 1

[0118] An arginine and oleanolic acid suspension comprising 0.026 g arginine, 0.034 g oleanolic acid, and 1 mL water.

[0119] A method for preparing an arginine and oleanolic acid suspension includes the following steps:

[0120] Arginine and oleanolic acid were added to water and stirred until homogeneous to obtain an arginine and oleanolic acid suspension.

[0121] Since the drug in Comparative Example 1 is in a particulate suspension state, the drug particles are difficult to disperse evenly in water or in the matrix of the needle tip, so it is difficult to prepare ideal microneedles. However, the co-amorphous composition formed by arginine and oleanolic acid of the present invention can self-assemble into a gel in water, which can be prepared as microneedles.

[0122] Performance testing

[0123] 1. X-ray diffraction analysis

[0124] X-ray diffraction analysis was performed on the hair loss treatment composition prepared in Example 1, and the results are as follows: Figure 3 As shown, the horizontal axis 2θ (degree) represents the diffraction angle 2θ (°).

[0125] Depend on Figure 3 As can be seen, no sharp diffraction peaks appeared on the X-ray diffraction pattern of the hair loss treatment composition prepared in Example 1, indicating the successful preparation of the co-amorphous material.

[0126] 2. Observation by transmission electron microscopy

[0127] The hydrogel prepared in Example 4 was observed by transmission electron microscopy, and the results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the hydrogel prepared in Example 4 has a layered cross structure.

[0128] 3. Rheological characteristics of hydrogels

[0129] The hydrogel formed by the self-assembly of the co-amorphous material in Example 1, i.e., the hydrogel in Example 4, was examined using amplitude and frequency scanning with an MCR 302e rheometer to investigate its rheological properties. The specific methods are as follows:

[0130] (1) Shear rate scan: The temperature is set to 32℃ and the initial shear rate is 1s. -1 The final shear rate is 100 s. -1 The viscosity at different shear rates was recorded to obtain the viscosity curve of the hydrogel.

[0131] (2) Amplitude scanning: Using a parallel plate fixture with a diameter of 20 mm, the temperature is set to 32℃, with an angular frequency of 10 rad / s and an amplitude variation range of 0.1-100%, the storage modulus (G') and loss modulus (G”) of the sample are measured to determine the linear viscoelastic region of the sample.

[0132] (3) Frequency scanning: Based on the range of the linear viscoelastic region, the strain is selected as 0.5%. A parallel plate fixture with a diameter of 25 mm is used, and the temperature is set to 32℃. Frequency scanning is performed in the range of 1-100 rad / s to measure the physical stability of the sample.

[0133] The rheological characteristics of the hydrogel in Example 4 are as follows: Figure 5 As shown, where, Figure 5 Figure (a) shows the viscosity curve of the hydrogel; Figure 5 Figure (b) shows the amplitude scanning results of the hydrogel; Figure 5 Figure (c) shows the angular frequency scan results of the hydrogel; Figure 5 In Figures (b) and (c), the vertical axis G'G" represents the energy storage modulus and the loss modulus, respectively. In Figures (b) and (c), G' represents the energy storage modulus and G" represents the loss modulus.

[0134] Depend on Figure 5 The viscosity curves show that the hydrogel has shear-thinning properties. The amplitude and angular frequency scans also show that the hydrogel has good elastic properties and a relatively stable structure.

[0135] 4. Characterization of the mechanical properties and separation properties of microneedles

[0136] The mechanical properties, sealing film porosity, and microneedle separation properties of the microneedles prepared in Case 1 were tested, and the specific methods are as follows:

[0137] (1) Mechanical performance testing: The mechanical strength of the microneedles was determined using a texture analyzer. The microneedles were placed on the stage with the tip facing upwards. The probe was moved to the tip of the microneedles, and the initial position was recorded. The probe was then moved downwards at a speed of 1 mm / s from the initial position. The force-displacement curves of the probe from the initial position until it completely crushed the microneedle were recorded. The mechanical performance results of the microneedles are as follows: Figure 6 As shown;

[0138] (2) Porosity test of sealing film: The insertion depth of the microneedles was determined using Parafilm sealing film. Each layer of sealing film was equivalent to the thickness of 100 μm of skin. Ten layers of sealing film were stacked to simulate human skin. The microneedles were placed on the sealing film with the needle tip facing down. A force of 30 N was applied vertically downwards to the backing layer of the microneedles using a digital push-pull force gauge. After maintaining this force for 1 minute, the microneedles were removed, and the porosity of each layer of sealing film was calculated. The porosity results of the microneedle sealing film were as follows: Figure 7 As shown;

[0139] (3) Microneedle dissociation test: Rhodamine B was loaded onto the tip of the microneedle to examine its skin puncture performance and drug delivery capability. The microneedle was placed tip-down on pigskin, and a digital push-pull force gauge was used to vertically insert the microneedle into the skin with a force of 30 N. After pressing for 1 minute, the microneedle was removed. Subsequently, the pigskin was frozen sectioned and photographed under a fluorescence microscope. The results of the microneedle dissociation test are as follows: Figure 8 As shown.

[0140] Figure 8 Figure (a) shows the appearance of pigskin before microneedle insertion. Figure 8 Image (b) shows the appearance of pigskin after microneedle insertion. Figure 8 Figure (c) shows the retention of microneedle tips in a frozen section.

[0141] Depend on Figure 6 It can be seen that the microneedle did not break during the stress process, and has good mechanical properties.

[0142] Depend on Figure 7 It is known that microneedles can penetrate to a depth of about 500 μm under the skin.

[0143] Depend on Figure 8 It is known that microneedles can effectively puncture the skin and achieve rapid separation of the needle tip and backing layer within 1 minute, allowing the needle tip to remain in the skin (fluorescence is added to the needle tip). Figure 8 (In Figure (c), the orange-red part represents the retained needle tip).

[0144] 5. In vitro drug release experiment of microneedles

[0145] The microneedles prepared in Application Example 1 were used to investigate the release characteristics of oleanolic acid from the microneedles.

[0146] This experiment used the Franz diffusion cell method to investigate the drug release performance. A 0.45 μm organic nylon filter membrane was used as an artificial membrane to simulate skin. Before use, the filter membrane was soaked in the release medium for 30 minutes. After fixing the device, the microneedles were placed in the supply cell and 100 μL of PBS was added. PBS containing 2% oleyl alcohol polyether (Oleth-20) was selected as the receiving solution. The receiving solution was added to the receiving cell, and the diffusion cell was placed in the diffusion apparatus. The temperature was set to 32℃ and the rotation speed was 250 rpm. 1 mL of release medium was taken at 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 120 h, and 1 mL of blank release medium was added. The samples were filtered through a 0.22 μm microporous filter membrane, and the content of oleanolic acid was determined by high performance liquid chromatography. The cumulative release amount at each time point was calculated according to formula (1), and the cumulative release rate (%) was calculated according to the ratio of the cumulative release amount to the total content of oleanolic acid in the microneedles.

[0147]

[0148] Where Cn is the drug concentration measured in the nth sampling, Ci is the drug concentration measured in each sampling, V represents the volume of the release medium, Vi represents the volume of each sampling, and Qn(μg) is the cumulative drug release.

[0149] Chromatographic conditions: XB-C18 column (250×4.6mm, 5μm); mobile phase: 0.1% phosphoric acid water: acetonitrile = 10:90 (v / v); flow rate: 1mL / min; detection wavelength: 210nm; injection volume: 20μL.

[0150] The in vitro drug release results of oleanolic acid in microneedles are as follows: Figure 9 As shown. By Figure 9 It can be seen that oleanolic acid exhibits a sustained-release trend in vitro in microneedles, and can continuously release the drug for 5 days. At 3 days, the release rate of oleanolic acid is 82.66±5.89%, and at 5 days, the release rate can reach 94.48±4.38%, which is basically complete.

[0151] 6. In vitro transdermal experiments of microneedles

[0152] The microneedles prepared in Application Example 1 were used to investigate the in vitro transdermal properties of oleanolic acid. The specific method is as follows:

[0153] Frozen rat skin was thawed by soaking in physiological saline, and the surface moisture was absorbed with filter paper. The integrity of the skin was then checked. The microneedles were pressed onto the stratum corneum of the skin with a 30N force using a digital push-pull force gauge and held for 3 minutes before being secured with medical 3M tape. Rat skin (stratum corneum facing the supply pool) and a pad were placed on top of the supply pool, and the receiving pool was secured. Phosphate-buffered saline (PBS) containing 2% oleth-20 was used as the receiving solution. The receiving solution was added to the receiving pool, and the diffusion pool was placed in a diffusion apparatus with the temperature set at 32℃ and the rotation speed at 250 rpm. Example 4 and Comparative Example 1 were selected as controls and added to the supply pool to maintain the same dosage per unit area as the microneedle group. 1 mL of receiving liquid was taken at 1, 2, 4, 8, 12, and 24 hours, and 1 mL of blank receiving liquid was added to ensure consistent volume of receiving liquid in the receiving pool. The obtained sample was filtered through a 0.22 μm microporous membrane, and the content of oleanolic acid was determined by high performance liquid chromatography. The cumulative permeate flow rate Qn per unit area of ​​oleanolic acid was calculated according to formula (2).

[0154]

[0155] Where A is the transdermal area, Cn is the measured drug concentration in the nth sampling, V is the volume of the diffusion medium, Vi is the volume of each sampling, Ci is the concentration measured in each sampling, and Qn is the cumulative permeation of the drug per unit area.

[0156] After the in vitro transdermal experiment, rat skin was removed from the diffusion cell. The skin at the drug administration site was wiped three times each with cotton balls soaked in water, methanol, and water to remove residual drug. The skin at the drug administration site was cut off, dried, placed in an EP tube, and then cut into pieces. The skin mass was weighed. Then, 1 mL of methanol was added to the EP tube, and the drug in the skin was extracted by sonication for 30 min. The mixture was then allowed to stand at room temperature for 2 h. The supernatant was filtered through a 0.22 μm microporous membrane, and the content of oleanolic acid was determined by high performance liquid chromatography, which is the skin retention amount.

[0157] The in vitro transdermal results of hydrogel in Example 4, microneedles in Application Example 1, and oleanolic acid in suspension in Comparative Example 1 are shown in Table 1.

[0158] Table 1: In vitro transdermal results of hydrogel in Example 4, microneedles in Application Example 1, and oleanolic acid in suspension in Comparative Example 1.

[0159]

[0160] Note: *** For a statistically significant difference to be found between this example and Comparative Example 1 (P < 0.001), ## For a statistically significant difference to be found between Example 4 and Example 5 (P<0.01), ### There was a statistically significant difference between this example and Example 4 (P<0.001).

[0161] As shown in Table 1, compared with the hydrogel of Example 4, the microneedles prepared in this invention can significantly enhance the skin penetration and retention of oleanolic acid, which is beneficial for oleanolic acid to exert its therapeutic effect on hair loss. For Comparative Example 1, because it is a suspension obtained by simply and directly mixing arginine and oleanolic acid, a co-amorphous system was not formed, resulting in undetectable skin penetration of oleanolic acid. Furthermore, the skin retention was significantly lower than in Example 4, and even lower than the microneedles used in Application Example 1. In other words, the skin penetration and retention of oleanolic acid in Comparative Example 1 were both poor, and it could not exert the therapeutic effect of oleanolic acid on hair loss.

[0162] 7. Cellular pharmacodynamic evaluation of arginine-oleanolic acid co-amorphous compounds

[0163] (1) Cell proliferation experiment

[0164] Human dermal follicular papillary cells (HDPCs) play a crucial role in regulating the hair follicle cycle. They promote hair follicle growth through division and proliferation, and lead to hair follicle regression by ceasing proliferation, remaining dormant during the telogen phase. Furthermore, they participate in hair follicle regeneration and repair processes. In summary, the proliferation and differentiation of HDPCs are of great significance for hair regeneration. Therefore, this study will investigate the effect of the arginine-oleanolic acid co-amorphous compound from Example 1 on the proliferative activity of HDPCs. The specific methods are as follows:

[0165] HDPC cells in the logarithmic growth phase were digested and seeded into 96-well plates at a density of 8000 cells / well. The plates were then incubated overnight. Arginine, oleanolic acid, and the co-amorphous compound prepared in Example 1 (i.e., the composition for treating hair loss) were diluted with complete DMEM medium to a concentration of 0.17-8.00 μg / mL and co-incubated with HDPC cells for 24 h. The 96-well plates were then removed from the incubator, the original medium was removed, and 120 μL of MTT diluent (containing 3-(4, 5-Dimethylthiazol-2-(2,5-diphenyltetrazolium bromide) (MTT 0.83 mg / mL) was added, and then the 96-well plate was wrapped with aluminum foil and placed in an incubator for incubation in the dark. After 4 hours, the culture medium in the wells was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was then shaken for 15-20 minutes to allow the crystals to fully develop. The entire process was carried out in the dark. Finally, the absorbance (OD) of each well in the 96-well plate was measured at 490 nm using a microplate reader, and the cell viability was calculated using the following formula:

[0166] Cell viability / % = (OD value of drug-treated group - OD value of apoptosis group) / (OD value of control group - OD value of apoptosis group) × 100%.

[0167] Among them, the treatment group was the group that both seeded cells and administered medication, the control group was the group that seeded cells but did not administer medication, and the zeroing group was the group that neither seeded cells nor administered medication.

[0168] The effects of the composition of Example 1, arginine, and oleanolic acid on the proliferation activity of HDPC are as follows: Figure 10 As shown in the figure. The concentrations in the first row of the horizontal axis represent the three concentrations of amorphous compounds, the concentrations in the second row represent the three concentrations of arginine, and the concentrations in the third row represent the three concentrations of oleanolic acid. ＊ This indicates a statistically significant difference compared to the control group (P<0.05). ＊＊ This indicates a statistically significant difference compared to the control group (P<0.01). ＊＊＊ This indicates a statistically significant difference compared to the control group (P<0.001). ## This indicates a statistically significant difference compared to the co-amorphous material (P<0.01).

[0169] Depend on Figure 10 It can be seen that at a concentration of 0.40 μg / mL, the co-amorphous compound significantly promoted the proliferation of HDPC cells. Arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL) at corresponding concentrations also had some proliferative effects, but their proliferation effects were not as good as those of the co-amorphous compound. This indicates that preparing arginine and oleanolic acid into a co-amorphous form not only does not affect their individual efficacy but also makes the proliferation effect of the combination superior to that of arginine and oleanolic acid used alone. When the concentration of the co-amorphous compound was 8.00 μg / mL, oleanolic acid at the corresponding concentration (4.6 μg / mL) had a certain toxic effect on cells, while the co-amorphous compound did not. This indicates that the co-amorphous compound can reduce the toxicity of oleanolic acid, further demonstrating the advantage of the combined use of the two drugs.

[0170] (2) Cell PCR experiment

[0171] To investigate whether the arginine-oleanolic acid co-amorphous compound can regulate the expression of Wnt / β-catenin and growth factors, which are related to hair growth, HDPC cells were modeled with dihydrotestosterone (DHT). After drug administration, real-time quantitative PCR (qRT-PCR) experiments were performed to examine the expression of mRNAs of CTNNBIP1, DKK1, TGFB1, and IGF1.

[0172] The specific method is as follows: HDPC cells in the logarithmic growth phase are digested and seeded into 12-well plates at a seeding density of 3 × 10⁶ cells / well. 5 Cells were cultured overnight at 100 cells / well to allow them to adhere. The original culture medium was then removed. DMEM basal medium was added to the control group, 1 μM DHT solution was added to the model group, and the remaining drug-treated groups were added with drug solution and 1 μM DHT solution, respectively. The cells were incubated for 24 hours. The drug-treated groups were arginine, oleanolic acid, and co-amorphous drug, respectively. The dosage of co-amorphous drug was 0.40 μg / mL. The dosage of single drug and corresponding drugs in co-amorphous drugs were consistent (arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL)). Subsequently, the cells were treated according to the PCR kit instructions, and fluorescence quantification was performed using a real-time fluorescence quantitative PCR instrument.

[0173] The relative expression level of CTNNBIP1 mRNA in human dermal follicular papillary cells is as follows: Figure 11 As shown, where, ＊ This indicates a statistically significant difference compared to the model group (P<0.05). ＊＊＊ This indicates a statistically significant difference compared to the model group (P<0.001). # This indicates a statistically significant difference compared to the amorphous group (P<0.05). ##This indicates a statistically significant difference compared to the co-amorphic group (P<0.01).

[0174] The relative expression level of DKK1 mRNA in human dermal follicular papillary cells is as follows: Figure 12 As shown. Among them, ＊＊＊ This indicates a statistically significant difference compared to the model group (P<0.001). ## This indicates a statistically significant difference compared to the amorphous group (P<0.01). ### This indicates a statistically significant difference compared to the co-amorphic group (P<0.001).

[0175] The relative expression level of TGFB1 mRNA in human dermal hair follicle papillary cells is as follows: Figure 13 As shown. Among them, ＊ This indicates a statistically significant difference compared to the model group (P<0.05). ＊＊＊ This indicates a statistically significant difference compared to the model group (P<0.001). ## This indicates a statistically significant difference compared to the co-amorphic group (P<0.01).

[0176] The relative expression level of IGF1 mRNA in human dermal hair follicle papillary cells is as follows: Figure 14 As shown. Among them, ＊＊ This indicates a statistically significant difference compared to the model group (P<0.01). ＊＊＊ This indicates a statistically significant difference compared to the model group (P<0.001). ## This indicates a statistically significant difference compared to the amorphous group (P<0.01). ### This indicates a statistically significant difference compared to the co-amorphic group (P<0.001).

[0177] Depend on Figure 11-14 It can be seen that the arginine-oleanolic acid co-amorphous compound prepared in Example 1, after acting on human dermal follicular papillary cells, can reverse the effects of dihydrotestosterone on the expression of various factors in HDPC cells. Specifically, it upregulates the mRNA expression of positive factors related to hair regeneration, such as CTNNBIP1 and IGF1, and downregulates the mRNA expression of negative factors, such as DKK1 and TGFB1. Furthermore, the above-mentioned effects of the arginine-oleanolic acid co-amorphous compound prepared in Example 1 are superior to those of the single-drug groups (arginine group and oleanolic acid group) at the corresponding concentrations, indicating that the arginine-oleanolic acid co-amorphous compound prepared in Example 1 has a better effect on regulating hair growth.

[0178] (3) Antioxidant experiment

[0179] Hair loss is closely related to vascular insufficiency and oxidative stress. Oxidative stress is mainly induced by excessive reactive oxygen species (ROS), leading to apoptosis of human dermal hair follicle papillary cells and weakening the ability of hair follicles to transition from the resting phase to the growth phase. This experiment investigated the antioxidant capacity of arginine, oleanolic acid, and the arginine-oleanolic acid co-amorphous compound prepared in Example 1 through antioxidant damage experiments and ROS scavenging experiments.

[0180] Antioxidant damage experiment: H2O2 was used to create a cell oxidative damage model to simulate the condition of human dermal hair follicle papillary cells under oxidative stress. Specifically, HDPC cells in the logarithmic growth phase were digested and seeded into 96-well plates at a seeding density of 8000 cells / well. The plates were then placed in an incubator for overnight culture. After cell adhesion, the original culture medium was removed, and different drug treatments were performed. The control group received only basic DMEM culture medium without any drugs. The model group received 600 μM H2O2. The drug treatment groups received arginine, oleanolic acid, and the arginine-oleanolic acid co-amorphous compound prepared in Example 1, in addition to H2O2. The dosage of the co-amorphous drug was 0.40 μg / mL, and the dosage of the single drug and the corresponding drug in the co-amorphous compound were consistent (arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL)). After 24 hours of drug incubation, cell viability was detected by MTT assay to understand the protective ability of different drugs against oxidative damage to cells.

[0181] The survival rates of human dermal follicular papillary cells in each treatment group under hydrogen peroxide conditions are as follows: Figure 15 As shown. Among them, ＊＊＊ This indicates a statistically significant difference compared to the model group (P<0.001). ### This indicates a statistically significant difference compared to the co-amorphic group (P<0.001).

[0182] Depend on Figure 15 It can be seen that the cell survival rate in the model group was significantly reduced, indicating a decrease in cell viability under oxidative stress. Compared with the model group, arginine, oleanolic acid, and the co-amorphous group all improved cell survival rate, indicating that all three have the ability to resist oxidative damage. Among them, the arginine-oleanolic acid co-amorphous compound group had the best protective effect on human dermal follicular papillary cells, with a cell survival rate comparable to the control group. This indicates that the arginine-oleanolic acid co-amorphous drug prepared from arginine and oleanolic acid can improve the survival rate of human dermal follicular papillary cells, enhance antioxidant capacity, and thus improve the prevention and treatment effects on hair loss.

[0183] ROS clearance assay: HDPC cells in logarithmic growth phase were digested and seeded in 12-well plates at a density of 1 × 10⁶ cells / well. 5Cells were cultured overnight in an incubator at 100 cells / well. After adhesion, the original culture medium was removed, and different drug treatments were applied. The control group was treated with only basic DMEM medium without any drugs. The model group was treated with 600 μM H2O2. The drug treatment groups were treated with arginine, oleanolic acid, and the arginine-oleanolic acid co-amorphous compound prepared in Example 1, respectively, in addition to H2O2. The dosage of the co-amorphous drug prepared in Example 1 was 0.40 μg / mL. The dosage of the single drug and the corresponding drug in the co-amorphous compound were consistent (arginine (0.17 μg / mL) and oleanolic acid (0.23 μg / mL)). After incubation for 24 hours, the original culture medium was removed, and the cells were washed three times with PBS. The cells were then co-incubated with the diluted ROS fluorescent probe DCFH-DA for 1 hour, followed by three more washes with PBS to remove any fluorescent probes that did not enter the cells. Finally, the cells were photographed using a fluorescence microscope to evaluate the drug's ability to scavenge ROS.

[0184] The ROS fluorescence detection results of human dermal follicular papillary cells in each treatment group under hydrogen peroxide conditions are as follows: Figure 16 As shown. Among them, Figure 16 The top-middle row shows images of cells from each treatment group under a fluorescence microscope. Since reactive oxygen species (ROS) are labeled, ROS expression can be visualized. Figure 16 The bottom row shows images of cells from each treatment group in bright field. Furthermore, the scale bars for all images are the same as those in the bright field images of the amorphous group, both at 200 μm.

[0185] Depend on Figure 16 It can be seen that both arginine and oleanolic acid have the activity of scavenging excessive ROS produced by cells due to oxidative damage, and the arginine-oleanolic acid co-amorphous compound formed by arginine and oleanolic acid has a better ROS scavenging ability than either drug alone (arginine or oleanolic acid). This indicates that the preparation of arginine-oleanolic acid co-amorphous drug from arginine and oleanolic acid can improve the ability to scavenge ROS, thereby improving the prevention and treatment effects on hair loss.

[0186] (4) Cell migration experiment

[0187] Vasification around hair follicles is crucial for maintaining healthy hair growth. Insufficient vascularization restricts the delivery of nutrients, biological factors, and immune cells to the hair follicle, affecting the hair follicle cycle and the repair of damaged hair follicles. Human umbilical vein endothelial cells (HUVECs) are a commonly used model cell for studying angiogenesis. Their potential for participating in angiogenesis can be assessed by detecting their proliferation and migration abilities. This experiment will examine the ability of the arginine-oleanolic acid co-amorphous compound prepared in Example 1 to promote HUVEC cell migration. The specific methods are as follows:

[0188] HUVEC cells were spaced at 5 × 10⁶ cells per well.5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. Using a 200 μL sterile pipette tip, cells were streaked perpendicularly along the bottom edge of the 6-well plate. The culture medium was aspirated, and the cells were washed 2-3 times with sterile PBS to remove the streaked cells. Arginine, oleanolic acid, and arginine-oleanolic acid co-amorphous compounds were diluted with incomplete DMEM medium to concentrations of 0.34 μg / mL, 0.46 μg / mL, and 0.80 μg / mL, respectively, and co-incubated with the cells. A control group was prepared with only basal DMEM medium. Cell migration was observed under a microscope at 0 h, 24 h, and 48 h after drug incubation. The scratch area at different time points was calculated using ImageJ software. The scratch area at 0 h was defined as the initial scratch area. Cell migration rate was calculated as follows:

[0189] Cell migration rate / % = (initial scratch area - scratch area) / initial scratch area × 100%.

[0190] The promoting effects of each treatment group on the migration of human umbilical vein endothelial cells are as follows: Figure 17 As shown, the scale of each figure is the same as the scale during the 48-hour incubation of the amorphous group.

[0191] The statistical results of the migration area of ​​human umbilical vein endothelial cells in each treatment group are as follows: Figure 18 As shown. Among them, ＊ This indicates a statistically significant difference compared to the control group (P<0.05). ＊＊ This indicates a statistically significant difference compared to the control group (P<0.01). ＊＊＊ This indicates a statistically significant difference compared to the control group (P<0.001). ## This indicates a statistically significant difference compared to the amorphous group (P<0.01). ### This indicates a statistically significant difference compared to the co-amorphic group (P<0.001).

[0192] Depend on Figure 17 and Figure 18 As can be seen, compared with the control group, both arginine and oleanolic acid have a certain promoting effect on the migration of HUVEC cells, and the arginine-oleanolic acid co-amorphous compound formed by the two drugs has a better promoting effect on migration than the arginine group and the oleanolic acid group treated alone. This indicates that the co-amorphous compound of the two drugs can help the formation of blood vessels around the hair follicles, providing nutritional conditions for hair growth.

[0193] In summary, the results of the cell experiments demonstrate that the co-amorphous compound composed of arginine and oleanolic acid can exert its therapeutic effect on hair loss through mechanisms such as promoting the proliferation of human dermal papilla cells, upregulating the Wnt / β-catenin pathway, improving oxidative stress around hair follicles, and addressing the microenvironment of insufficient angiogenesis. Compared with the use of arginine and oleanolic acid alone, the combined use does not affect the efficacy of the drugs while providing complementary effects in terms of therapeutic mechanisms.

[0194] 8. In vivo pharmacodynamic evaluation

[0195] A hair loss animal model was established using 7-week-old C57BL / 6 male mice. The control group mice were treated without any treatment after the hair on their backs was removed. The other groups of mice were treated with 0.1 mL of 0.5% testosterone solution applied topically to the hair removal area daily for 2 weeks. The testosterone solution was prepared with 50% ethanol. Except for the control group, the other mice were randomly divided into 5 groups (n=6). The administration methods for each group were as follows: (1) Control group: No treatment was given; (2) Model group: 0.1 mL of 0.5% testosterone solution was applied topically daily; (3) Co-amorphous gel group: Hydrogel from Example 4 was applied topically every 3 days; (4) Blank microneedle group: Blank microneedles without any drugs were applied once every 3 days; (5) Co-amorphous microneedle group: Co-amorphous microneedles from Example 1 were applied once every 3 days; (6) 5% minoxidil group: 0.1 mL of commercially available minoxidil tincture with a concentration of 5% was applied topically once daily. In all groups except the control group (1), 0.1 mL of 0.5% testosterone solution was applied topically daily. Groups (3)-(6) were administered the same solution 8 hours later. During the administration period, the hair regeneration of mice was recorded by photographing on days 0, 3, 6, 9, 12, 14, and 16. The hair coverage of the back skin of the mice was calculated using ImageJ software. After the experiment, the mice were euthanized by cervical dislocation, and the hair and skin on their backs were removed. The length of the regenerated hair was measured using calipers.

[0196] The following figures show the hair growth on the backs of mice in each group during the drug administration period. Figure 19 As shown.

[0197] The statistical graph of hair regeneration coverage rate in mice of each group after drug administration is shown below. Figure 20 As shown. Among them, ＊＊＊ This indicates a statistically significant difference compared to the amorphous microneedle group (P<0.001).

[0198] The statistical graph of hair regeneration length in mice of each group after drug administration is shown below. Figure 21 As shown. Among them, ＊＊＊ This indicates a statistically significant difference compared to the amorphous microneedle group (P<0.001).

[0199] Depend on Figure 19As can be seen, compared with the model group, all four drug-treated groups promoted hair growth in mice. Figure 20 and Figure 21 Statistical analysis of the hair regrowth coverage and length in each group of mice showed that the amorphous microneedle group and the 5% minoxidil tincture group had comparable effects in promoting hair growth, which were superior to the amorphous gel group and the blank microneedle group. The blank microneedle group, due to the skin stimulation after application, could increase blood supply to hair follicles to some extent, thus also promoting hair growth, but its effect was the worst among all treatment groups. Furthermore, the aforementioned transdermal experiment showed that the drug delivery efficiency of the amorphous gel group and the microneedle group loaded with amorphous drugs was much higher than that of Comparative Example 1; and since the drug in Comparative Example 1 was in suspension, it was difficult for the drug to penetrate the skin, so poor treatment effect was predictable. Therefore, this group was not designed in animal experiments to avoid wasting experimental animal resources.

[0200] In summary, the arginine and pentacyclic triterpenoids of this invention exhibit a synergistic effect, leveraging complementary therapeutic mechanisms compared to using the active ingredients alone, significantly enhancing the treatment efficacy for hair loss. Furthermore, the co-amorphous system formed by arginine and pentacyclic triterpenoids upregulates the Wnt / β-catenin pathway, improving the microenvironment surrounding hair follicles with oxidative stress and insufficient angiogenesis, providing favorable conditions for hair follicle growth and effectively preventing and treating hair loss. Moreover, further processing the co-amorphous system obtained from arginine and pentacyclic triterpenoids into microneedles enhances the therapeutic effect. Simultaneously, the separable design of the microneedles allows for effective drug retention and sustained release within the skin, thereby reducing the frequency of drug administration and providing a novel and effective strategy for hair loss treatment.

[0201] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The use of a composition for treating hair loss in the preparation of a medicament for treating hair loss, characterized in that, The composition comprises arginine and pentacyclic triterpenoids; the composition is a co-amorphous compound. The drug includes microneedles, which sequentially comprise a backing layer, a separation layer, and a needle tip; the needle tip is composed of a hydrogel. The hydrogel is formed by self-assembly of the composition; The pentacyclic triterpenoids include at least one of oleanolic acid, glycyrrhetinic acid, and ursolic acid.

2. The application according to claim 1, characterized in that, The molar ratio of arginine to pentacyclic triterpenoids is (0.9-4.5):

1.

3. The application according to claim 1, characterized in that, The method for preparing the composition includes the following steps: (1) Arginine and water are mixed to obtain an arginine solution; a pentacyclic triterpenoid compound, an alcohol and / or an ether are mixed to obtain a dispersion of the pentacyclic triterpenoid compound; (2) The arginine solution obtained in step (1) and the dispersion of the pentacyclic triterpenoid compound are mixed, ultrasonically treated, and dried to obtain the composition.

4. The application according to claim 1, characterized in that, The mass ratio of the backing layer, the separation layer, and the needle tip is (1.5-4.5):(0.8-1.2):

1.

5. The application according to claim 1, characterized in that, The method for preparing the microneedles includes the following steps: The needle tip, the separation layer, and the backing layer are sequentially formed in a mold to obtain the microneedle.

Images