An ionic liquid for improving androgenetic alopecia and its applications

Abstract

This invention belongs to the field of biomedical materials technology, specifically relating to an ionic liquid for improving androgenetic alopecia and its applications. The ionic liquid is prepared according to the following steps: L-L-carnitine and succinic acid are reacted under inert conditions to obtain an L-L-carnitine-succinic acid ionic liquid with an ion-hydrogen bond network structure; vanillin is added to the L-L-carnitine-succinic acid ionic liquid, and the mixture is heated and stirred to obtain the ionic liquid. This ionic liquid can function in the microenvironment of dermal papilla cells, improving the hair follicle microenvironment and enhancing the activity of dermal papilla cells, which play a key role in androgenetic alopecia. Furthermore, this drug-loaded ionic liquid can effectively inhibit androgen receptor activity, preventing the binding of androgens and androgen receptors from causing problems such as hair follicle miniaturization and hair loss. Simultaneously, it also has the effect of improving androgenetic alopecia and promoting hair growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an ionic liquid for improving androgenetic alopecia and its applications. Background Technology

[0002] Hair loss is a skin condition characterized by excessive or abnormal hair loss and a decrease in the number of hairs. Androgenetic alopecia (AGA) is one of the most common types of chronic hair loss, affecting up to 80% of men and 50% of women, and its incidence often increases with age. Androgenetic alopecia is related to genetics and hormone levels, with genetic susceptibility being the underlying cause. The sensitivity of hair follicles to androgens (especially dihydrotestosterone, DHT) is genetically determined and exhibits familial aggregation. The key to the effect of androgens lies not in the absolute level of androgens (many patients have normal hormone levels), but in the abnormal sensitivity of hair follicles to DHT. The treatment principles for androgenetic alopecia are early intervention, long-term adherence, and combination therapy. The goal is to slow the hair loss process and promote hair growth, but it is difficult to regenerate completely dead hair follicles.

[0003] Currently, the most common treatments for AGA are oral finasteride and topical minoxidil, but these medications have certain limitations. For example, minoxidil can cause skin irritation and itching, finasteride is not suitable for women, and long-term use in men may lead to sexual dysfunction. Therefore, there is an urgent need to develop new products that can effectively promote hair regeneration while also possessing good efficacy and biocompatibility. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an ionic liquid for improving androgenetic alopecia, addressing the shortcomings of the prior art.

[0005] The specific technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides an ionic liquid for improving androgenetic alopecia, which is prepared according to the following steps:

[0007] L-L-carnitine and succinic acid were reacted under inert conditions, and anions and cations interacted (Coulomb interaction) and hydrogen bonding to obtain an L-L-carnitine-succinic acid ionic liquid with an ionic-hydrogen bond network structure.

[0008] Vanillin was added to the L-carnitine-succinic acid ionic liquid, and the mixture was heated and stirred to allow vanillin to undergo non-covalent interaction with the ionic-hydrogen bond network structure, thus obtaining the ionic liquid.

[0009] In a preferred embodiment of the present invention, the molar ratio of L-carnitine to succinic acid is 1:1~3.

[0010] In a preferred embodiment of the present invention, L-L-carnitine and succinic acid powders are dissolved in an organic solvent for reaction, and the mass ratio of the sum of the masses of L-L-carnitine and succinic acid to the mass of the organic solvent is 1 mg: 10-20 L.

[0011] In a preferred embodiment of the present invention, the molar ratio of vanillin ethyl ketone to the L-carnitine-succinic acid ionic liquid is 1:2 to 8.

[0012] As a preferred embodiment of the present invention, vanillin and L-carnitine-succinate ionic liquid are dissolved in PBS, heated and stirred to obtain L-carnitine-succinate ionic liquid loaded with vanillin.

[0013] As a preferred embodiment of the present invention, the reaction of L-carnitine and succinic acid under inert conditions is carried out in a nitrogen atmosphere at 50°C to 80°C with a stirring speed of 200 rpm to 800 rpm for 24 h to 48 h.

[0014] In a preferred embodiment of the present invention, the heating and stirring temperature is 30-40°C, the stirring speed is 300-500 rpm, and the stirring time is 1-5 hours.

[0015] As a preferred embodiment of the present invention, the reaction product of L-carnitine and succinic acid is dried under vacuum at 60-80 °C to obtain an L-carnitine-succinic acid ionic liquid with an ion-hydrogen bond network structure.

[0016] In a second aspect, the present invention provides the application of the ionic liquid in the preparation of cosmetics for preventing hair loss and strengthening hair.

[0017] Among them, L-L-carnitine-succinate ionic liquid loaded with vanillin (Apo) has antioxidant, androgen receptor inhibition, and β-catenin / Wnt3a pathway activation effects.

[0018] In a preferred embodiment of the present invention, the ionic liquid is used for androgenetic alopecia.

[0019] In a third aspect, the present invention provides the use of the ionic liquid in the preparation of a drug that promotes hair growth.

[0020] In a fourth aspect, this invention provides a cosmetic for preventing hair loss and strengthening hair, which is formulated from the aforementioned ionic liquid and acceptable excipients. Acceptable excipients include, but are not limited to: surfactants (such as sodium lauroyl glutamate, sodium methyl cocoyl taurate, sodium laureth sulfate, sodium lauryl sulfate, cocamidopropyl betaine, cocamidopropyl MEA), alkyl glycosides, conditioning agents (such as polyquaternium-10, polyquaternium-7, polydimethylsiloxane, cyclopentamethoxysiloxane, natural oils and esters), thickeners (such as xanthan gum, carbomer, hydroxyethyl cellulose), pH adjusters (citric acid, lactic acid, sodium hydroxide, triethanolamine, etc.), preservatives (such as phenoxyethanol, capryloyl hydroxamic acid, p-hydroxyacetophenone, ethylhexylglycerin, sodium benzoate, potassium sorbate), penetration enhancers (such as azone, ethyl nicotinate), antioxidants (such as vitamin E, disodium EDTA), etc.

[0021] In a fifth aspect, the present invention provides a medicament for promoting hair growth, which is formulated from the ionic liquid and acceptable excipients. Acceptable excipients include, but are not limited to: solubilizers (such as propylene glycol, polyethylene glycol), penetration enhancers (such as ethanol, propylene glycol, azone), antioxidants (such as sodium metabisulfite, disodium EDTA), pH adjusters (such as citric acid, hydrochloric acid, sodium hydroxide), colorants, etc.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention provides an ionic liquid for improving androgenetic alopecia, wherein vanillin (Apo), L-carnitine (LC), and succinic acid (SA) respectively perform three functions in the system: drug loading activity, ionic liquid cation / anion pairing, and regulation of the hair follicle microenvironment, thereby producing synergistic effects at the following levels:

[0024] Firstly, carrier-level synergy (dissolution and delivery): The ionic liquid (CSIL) composed of LC / SA interacts non-covalently with Apo through an ion-hydrogen bond network, improving the dispersion and usability of Apo in aqueous phase / PBS, overcoming its hydrophobic limitation, and ensuring its effective exposure in the microenvironment of dermal papilla cells (HFDPC).

[0025] Secondly, synergistic effects at the microenvironment level (antioxidant → metabolic homeostasis): Apo reduces the ROS content in the microenvironment by inhibiting NADPH oxidase; LC and SA in the CSIL component participate in fatty acid mitochondrial transport and tricarboxylic acid cycle, respectively, promoting cellular energy metabolism homeostasis. The combination of these two with Apo achieves bidirectional regulation of reducing ROS and promoting cellular metabolism, thus improving the hair follicle microenvironment.

[0026] Third, target / pathway synergy (AR and Wnt pathways): In the DHT-induced model, the combination of Apo and CSIL showed a stronger inhibitory effect on androgen receptor (AR) activity than the single component, and was accompanied by activation of the β-catenin / Wnt3a signaling pathway, thereby promoting dermal papilla cell migration and angiogenesis.

[0027] Fourth, the integration effect: the above-mentioned multi-level synergy ultimately manifests as a significant increase in hair coverage in the mouse AGA model, superior to Apo or CSIL alone. See also Figures 4-8 Results and comparisons.

[0028] The drug-loaded ionic liquid provided by this invention not only effectively improves the water solubility of vanillin but also effectively inhibits androgen receptors, preventing 5α-reductase from catalyzing the conversion of testosterone to DHT. This avoids the problems caused by DHT binding to androgen receptors and leading to rapid entry into the regression phase, miniaturization, and hair loss in hair follicles. Simultaneously, this drug-loaded ionic liquid can also inhibit the production of reactive oxygen species in the skin's hair follicles, achieving the purpose of preventing hair loss and promoting hair growth. Attached Figure Description

[0029] Figure 1 The image shows the physical sample of the prepared L-L-carnitine-succinic acid ionic liquid.

[0030] Figure 2 TEM image of the prepared L-L-carnitine-succinic acid ionic liquid.

[0031] Figure 3 To assess the blood compatibility of different samples.

[0032] Figure 4 The scavenging rate of hydroxyl radicals for different samples is shown.

[0033] Figure 5 The effect of different concentrations of samples on the migration rate of dermal papilla cells. A. Changes in cell migration; B. Statistical graph of cell migration rate.

[0034] Figure 6 The effect of different concentrations of samples on the angiogenesis rate of human umbilical vein endothelial cells. A. Total vessel length, B. Vessel junctions, C. Tube formation images of HUVEC cells after different treatments.

[0035] Figure 7 The effect of different samples on AR.

[0036] Figure 8 This is a schematic diagram showing the hair coverage of mice during the treatment phase. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples.

[0038] This invention provides an L-L-carnitine-succinic acid ionic liquid loaded with vanillin, which is prepared according to the following steps:

[0039] Step 1: Dissolve L-L-carnitine and succinic acid powder in an organic solvent.

[0040] Succinic acid was added at a ratio of 1:1 to 3, and the mixture was stirred at 50 to 80 °C and 200 to 800 rpm for 24 to 48 h under a nitrogen atmosphere. The mixture was then vacuum dried at 60 to 80 °C to obtain L-L-carnitine succinic acid ionic liquid.

[0041] Step 2: Dissolve Apo and L-carnitine-succinate ionic liquid in ethanol and stir at 30-40 °C and 300-500 rpm to obtain L-carnitine-succinate ionic liquid loaded with vanillin.

[0042] Apocynin is a natural product with broad biological activity and is a natural NADPH oxidase inhibitor. However, the physicochemical properties of apocynin show that it is insoluble in water at room temperature and its molecular structure is highly hydrophobic. This poor water solubility limits its application in improving androgenetic alopecia. L-L-carnitine-succinic acid ionic liquid can effectively improve the solubility of apocynin. Furthermore, L-L-carnitine-succinic acid ionic liquid also promotes hair growth. Therefore, the L-L-carnitine-succinic acid ionic liquid prepared in this invention can not only serve as a carrier for apocynin but also synergistically work with apocynin to improve androgenetic alopecia.

[0043] This ionic liquid can function within the microenvironment of dermal papilla cells, improving the hair follicle microenvironment and enhancing the activity of dermal papilla cells, which play a crucial role in androgenetic alopecia. Furthermore, this drug-loaded ionic liquid effectively inhibits androgen receptor activity, preventing androgens from binding to androgen receptors and causing hair follicles to rapidly enter the regression phase, leading to follicle miniaturization and hair loss. Simultaneously, it can inhibit ROS production in skin hair follicles, promote angiogenesis, and activate the β-catenin / Wnt3a pathway, thereby improving androgenetic alopecia and promoting hair growth.

[0044] Example 1

[0045] An L-L-carnitine-succinic acid ionic liquid loaded with vanillin was prepared according to the following steps:

[0046] Step 1: Dissolve L-L-carnitine and succinic acid powder in ethanol. The L-L-carnitine and succinic acid are added at a molar ratio of 1:3, and the material-to-liquid ratio is 1 mg: 10 L. Under a nitrogen atmosphere, the mixture is heated at 50 °C and stirred at 500 rpm for 24 h. Finally, the ethanol is removed by vacuum drying at 60 °C to obtain a light yellow viscous L-L-carnitine succinic acid ionic liquid.

[0047] Step 2: Dissolve vanillin ethyl ketone and L-L-carnitine-succinic acid ionic liquid in ethanol at a molar ratio of 1:4. Heat at 35 °C and stir at 500 rpm for 4 h to obtain L-L-carnitine-succinic acid ionic liquid loaded with vanillin ethyl ketone.

[0048] Example 2

[0049] An L-L-carnitine-succinic acid ionic liquid loaded with vanillin was prepared according to the following steps:

[0050] Step 1: Dissolve L-L-carnitine and succinic acid powder in ethanol. The L-L-carnitine and succinic acid are added at a molar ratio of 1:3, with a material-to-liquid ratio of 1 mg: 20 L. Under a nitrogen atmosphere, the mixture is heated at 60 °C and stirred at 500 rpm for 36 h. Finally, the ethanol is removed by vacuum drying at 70 °C to obtain a pale yellow viscous L-L-carnitine-succinic acid ionic liquid.

[0051] Step 2: Dissolve vanillin ethyl ketone and L-L-carnitine-succinic acid ionic liquid in ethanol at a molar ratio of 1:2. Heat at 40 °C and stir at 300 rpm for 5 h to obtain L-L-carnitine-succinic acid ionic liquid loaded with vanillin ethyl ketone.

[0052] Example 3

[0053] An L-L-carnitine-succinic acid ionic liquid loaded with vanillin was prepared according to the following steps:

[0054] Step 1: Dissolve L-L-carnitine and succinic acid powder in ethanol. Add L-L-carnitine and succinic acid at a molar ratio of 1:2, with a material-to-liquid ratio of 1 mg:10 L. Heat at 50 °C and stir at 800 rpm for 24 h under a nitrogen atmosphere. Finally, vacuum dry at 50 °C to obtain a light yellow viscous L-L-carnitine-succinic acid ionic liquid.

[0055] Step 2: Dissolve vanillin ethyl ketone and L-L-carnitine-succinic acid ionic liquid in ethanol at a molar ratio of 1:8. Heat at 30 °C and stir at 500 rpm for 1 h to obtain L-L-carnitine-succinic acid ionic liquid loaded with vanillin ethyl ketone.

[0056] Example 4

[0057] An L-L-carnitine-succinic acid ionic liquid loaded with vanillin was prepared according to the following steps:

[0058] Step 1: Dissolve L-L-carnitine and succinic acid powder in ethanol. Add L-L-carnitine and succinic acid at a molar ratio of 1:2, with a material-to-liquid ratio of 1 mg:10 L. Under a nitrogen atmosphere, heat at 70 °C and stir at 800 rpm for 48 h. Finally, vacuum dry at 70 °C to obtain a light yellow viscous L-L-carnitine and succinic acid ionic liquid.

[0059] Step 2: Vanillin ethyl ketone and L-L-carnitine-succinic acid ionic liquid were dissolved in ethanol at a molar ratio of 1:4. The mixture was heated at 30 °C and stirred at 500 rpm for 2 h to obtain L-L-carnitine-succinic acid ionic liquid loaded with vanillin ethyl ketone.

[0060] Example 5

[0061] An L-L-carnitine-succinic acid ionic liquid loaded with vanillin was prepared according to the following steps:

[0062] Step 1: Dissolve L-L-carnitine powder and succinic acid powder in ethanol. Add L-L-carnitine and succinic acid at a molar ratio of 1:1, with a material-to-liquid ratio of 1 mg:10 L. Under a nitrogen atmosphere, heat at 80 °C and stir at 800 rpm for 36 h. Finally, vacuum dry at 70 °C to remove water and obtain a white, viscous L-L-carnitine-succinic acid ionic liquid.

[0063] Step 2: Dissolve vanillin ethyl ketone and L-L-carnitine-succinic acid ionic liquid in ethanol at a molar ratio of 1:2. Heat at 37 °C and stir at 500 rpm for 4 h to obtain L-L-carnitine-succinic acid ionic liquid loaded with vanillin ethyl ketone.

[0064] Comparative Example 1

[0065] L-carnitine powder and succinic acid powder were dissolved in ethanol. L-carnitine and succinic acid were added at a molar ratio of 3:1, with a material-to-liquid ratio of 1 mg:10 L. The mixture was heated at 30 °C and stirred at 500 rpm for 24 h under a nitrogen atmosphere. Finally, the mixture was vacuum dried at 70 °C to remove water and obtain a white solid powder.

[0066] Experiments showed that ionic liquids could not be formed when the reaction temperature in step one was below 40°C. Comparative Example 1, with a molar ratio of 3:1, also failed to form an ionic liquid, ultimately yielding a white solid powder.

[0067] Comparative Example 2

[0068] L-L-carnitine and succinic acid powders were dissolved in ethanol at a molar ratio of 1:3 (1 mg: 20 L). The mixture was heated at 60 °C and stirred at 500 rpm for 36 h under a nitrogen atmosphere. Finally, the ethanol was removed by vacuum drying at 70 °C to obtain a pale yellow, viscous L-L-carnitine-succinic acid ionic liquid. Vanillin and the L-L-carnitine-succinic acid ionic liquid were dissolved in ethanol at a molar ratio of 1:2. The mixture was stirred at 600 rpm for 30 min at room temperature. However, vanillin was not fully loaded.

[0069] Experimental analysis: The experiment found that vanillin ethyl ketone could not be fully loaded when the reaction temperature in step two was less than 30 ℃ and the reaction time was less than 1 h.

[0070] Structural analysis of materials is performed using transmission electron microscopy (TEM). Figure 2 The results showed that the ionic liquid particles were around 100 nm in size and had a uniform morphology, which was consistent with the particle size test results.

[0071] Experimental Example 1

[0072] Biocompatibility testing

[0073] The blood compatibility of ionic liquids was assessed using an in vitro hemolysis assay. Sterile defibrinated rabbit blood was centrifuged at 1500 rpm for 10 min, and the lower red blood cells were collected and diluted with NaCl to a 5% red blood cell suspension. In this study, physiological saline was selected as the negative control and deionized water as the positive control. Briefly, 0.5 mL of vanillin-based ionic liquid (Apo), L-carnitine-succinate ionic liquid (CSIL), vanillin-loaded L-carnitine-succinate ionic liquid (A@CSIL), the negative control (0.9% NaCl), and 0.5 mL of the positive control (ultrapure water) were added to 2 mL centrifuge tubes. Then, 0.5 mL of freshly diluted blood was added to each tube, and the mixture was incubated in a 37 °C water bath for 1 h. After centrifugation at 1500 rpm for 5 min, the supernatant was transferred to a 96-well plate, and the absorbance was measured at 540 nm. The hemolysis rate was calculated using the following formula:

[0074]

[0075] Where As represents the absorbance of different samples, Ap represents the absorbance of deionized water, and An represents the absorbance of physiological saline.

[0076] Depend on Figure 3 It is known that the L-L-carnitine succinate ionic liquid loaded with vanillin has good biocompatibility.

[0077] Experiment Example 2

[0078] Hydroxyl radical (·OH) scavenging rate

[0079] A 2 mM FeSO4 and H2O2 solution was prepared, with a concentration ratio of FeSO4 to H2O2 of 1:1. Samples were divided into a blank control group, a vanillin group (Apo), an L-carnitine-succinate ionic liquid group (CSIL), and a vanillin-loaded L-carnitine-succinate ionic liquid group (A@CSIL). The concentrations of vanillin and L-carnitine-succinate ionic liquid in each group were 0.5 mg / mL and 1.58 mg / mL, respectively. Samples from different groups were added and reacted at 37 ℃ for 2 h. The reaction solution was then reacted with 3,3',5',5-tetramethylbenzidine at a volume ratio of 1:1 for the next step. After reacting for 15 min, each solution was transferred to a 96-well plate, and the absorbance at 650 nm was measured. The hydroxyl radical scavenging rate was calculated using the following formula:

[0080]

[0081] Where As represents the absorbance of different samples, An represents the absorbance of deionized water, and Ap represents the absorbance of the blank control group.

[0082] like Figure 4 The results showed that vanillin and L-carnitine-succinic acid ionic liquids had scavenging rates of 53.86% and 33.57% for hydroxyl radicals, respectively, which were not ideal. However, the vanillin-loaded L-carnitine-succinic acid ionic liquid achieved a scavenging rate of 86.18% for hydroxyl radicals, indicating that vanillin and L-carnitine-succinic acid ionic liquids can synergistically remove excess reactive oxygen species in the hair follicle microenvironment.

[0083] Experimental Example 3

[0084] Hair papilla cell migration rate detection experiment

[0085] Cell migration rate: The cell scratch assay is used to determine the cell migration ability. The experiment consists of four steps: streaking the culture plate, seeding cells, scratching cells, and observing cell healing. The specific operation steps are as follows:

[0086] a. Marking the culture plate: Place the 6-well plate upside down on the table and use a marker to draw horizontal lines evenly across the bottom of the plate with a ruler, approximately every 0.5 to 1 cm. Each well should have at least 3 lines.

[0087] b. Inoculate cells: Add 2 mL of cells, totaling approximately 5 × 10⁶ cells. 5 Cells were seeded in 6-well culture plates and cultured in a CO2 incubator at 37 ℃ until the cells adhered to the wells and covered the bottom of the plates.

[0088] c. Cell Scribing: Remove the culture plate and observe the cell state. Place a sterile steel ruler on the six-well plate and scribble a line in the center of the cells using a sterile 200 μL pipette tip, perpendicular to the horizontal line drawn in step a. Keep the pipette tip as straight as possible during scribing, avoiding any tilting. Discard the original culture medium and wash the cells three times with PBS to remove any remaining scribbled cells. Photograph the wells under a microscope and record the time as 0 h.

[0089] d. The experiment consisted of five groups: a blank control group, a DHT group, a vanillin group (Apo), an L-carnitine-succinate ionic liquid group (CSIL), and a vanillin-loaded L-carnitine-succinate ionic liquid group (A@CSIL). The blank control group received serum-free culture medium, while the other groups received samples diluted with serum-free medium. The concentrations of vanillin and L-carnitine-succinate ionic liquid in the samples were 332 ng / mL and 1 μg / mL, respectively. The culture plates were incubated at 37 ℃ in a 5% CO2 incubator for 12 h. Afterward, photographs were taken under a microscope (keeping the position consistent with 0 h) to observe the repair of cell scratches. The scratch area was analyzed using ImageJ software, and cell migration rate was calculated using the following formula:

[0090]

[0091] Where A0 and At are the initial scratch area and the scratch area after 0 h and 12 h of culture, respectively.

[0092] Cell culture and passage: Remove the cryopreserved tubes of dermal papilla cells from liquid nitrogen, incubate at 37°C with shaking, and after complete thawing, quickly add the cells to the primary fibroblast basal culture medium (containing 10% fetal bovine serum and 1×10⁻⁶ oz.) using a sterile pipette. 5 In a T25 cell culture flask containing U / L penicillin and 100 mg / L streptomycin, mix well using the cross-hatching method and then place the flask in a cell culture incubator at 37 ℃, 5% CO2, and saturated humidity. After observing cell adhesion for 4 hours, change the medium and continue culturing.

[0093] For cells with a density exceeding 80%, passage them: aspirate the cell culture medium, gently wash twice with PBS, add trypsin containing 0.25% EDTA, and digest in an incubator for 2 min. Terminate cell digestion with fresh primary fibroblast basal medium. Centrifuge the cell suspension at 1500 rpm for 5 min. Add fresh primary fibroblast basal medium and agitate the cell pellet. The number of passage flasks depends on the cell count, cell condition, and experimental requirements. Place the culture flasks in a cell culture incubator for further culture.

[0094] like Figure 5The results showed that both vanillin and L-carnitine-succinate ionic liquid had some effect on promoting cell migration in the DHT-constructed HFDPC cell model, but the effect of vanillin-loaded L-carnitine-succinate ionic liquid on promoting cell migration was more significant. Specifically, the cell migration rates in the normal group and the DHT-constructed cell model group were 44.73% and 20.22%, respectively; the cell migration rates in the vanillin group and the L-carnitine-succinate ionic liquid group were 34.92% and 45.34%, respectively, while the cell migration rate in the vanillin-loaded L-carnitine-succinate ionic liquid group was 50%. These results indicate that vanillin and L-carnitine-succinate ionic liquid can improve the inhibition of cell migration by DHT, restoring the cell migration rate to normal levels and synergistically promoting cell migration.

[0095] Experiment Example 4

[0096] Angiogenesis rate detection experiment

[0097] Human umbilical vein endothelial cell (HUVEC) angiogenesis assay: After HUVEC cells adhered to the plate for 24 h, the culture medium was aspirated, and the bottom of the plate was washed twice with PBS. 2 mL of freshly prepared drug was added to each well, with blank control and sample groups of different concentrations established, and the cells were cultured for another 48 h.

[0098] Cell culture and passage: Remove the HUVEC cell cryopreservation tubes from liquid nitrogen, incubate at 37°C with shaking, and after complete thawing of the cryopreservation solution, quickly add the cells to DMEM medium (containing 10% fetal bovine serum and 1×10⁻⁶ ppm) using a sterile pipette. 5 In a T25 cell culture flask containing U / L penicillin and 100 mg / L streptomycin, mix well using the cross-hatching method and then place the flask in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. After observing cell adhesion for 4 hours, change the medium and continue culturing.

[0099] For cells with a density exceeding 80%, passage them: aspirate the cell culture medium, gently wash twice with PBS, add trypsin containing 0.25% EDTA, and digest for 3 min in an incubator. Terminate cell digestion with fresh DMEM complete medium. Centrifuge the cell suspension at 1500 rpm for 5 min. Add fresh DMEM complete medium and agitate the cell pellet. The number of passage flasks depends on the cell count, cell condition, and experimental requirements. Place the culture flasks in a cell culture incubator for further culture.

[0100] like Figure 6The results showed that the normal group had 115 branching points and a total vessel length of 11524 μm; the DHT-constructed cell model group had 45 branching points and a total vessel length of 7253 μm; the vanillin-loaded group had 61 branching points and a total vessel length of 9000 μm; the L-carnitine-succinate ionic liquid group had 81 branching points and a total vessel length of 10215 μm; and the vanillin-loaded L-carnitine-succinate ionic liquid group had 94 branching points and a total vessel length of 10816 μm, with angiogenesis restored to normal levels. These results indicate that vanillin and L-carnitine-succinate ionic liquid have a synergistic effect on promoting angiogenesis in the DHT-constructed HFDPC cell model.

[0101] Experimental Example 5

[0102] Western blot (WB) assay to detect activation pathways

[0103] Western blot (WB) assays are used to detect the expression of key proteins in pathways to determine whether drugs regulate target pathways. The specific steps are as follows:

[0104] Step 1: Protein extraction, the specific steps are as follows:

[0105] 0. Take 6-well plates treated with different drugs, discard the culture medium, and quickly wash the cells 2-3 times with pre-cooled PBS;

[0106] 1. Add 150-200 μL of RIPA lysis buffer containing protease / phosphatase inhibitors to each well and lyse on ice for 30 min (during which time repeatedly pipette to ensure complete cell lysis);

[0107] Centrifuge at 2.4℃ and 12000 rpm for 15 min, then transfer the supernatant (i.e., protein sample) to a new centrifuge tube and discard the precipitate (cell debris).

[0108] 3. Detect protein concentration using the BCA or Bradford method (to avoid differences in sample loading amount). Dilute all samples to the same concentration (e.g., 2 μg / μL) with lysis buffer according to the quantitative results, then add 5×SDS loading buffer (final concentration 1×), boil at 95 ℃ for 5 min to denature the protein, and store at -20 ℃ for later use (short-term storage: 1 week; long-term storage: -80 ℃).

[0109] Step 2: SDS-PAGE gel electrophoresis

[0110] 4. Gel preparation: Prepare separating gel according to protein molecular weight, pour into the electrophoresis tank, insert comb, and let it solidify at room temperature for 30 minutes (avoid air bubbles, otherwise it will affect electrophoresis).

[0111] 5. Sample loading and electrophoresis: Add 1×SDS electrophoresis buffer to the electrophoresis tank, remove the comb, load 20-50 μg of protein sample into each well, and simultaneously load the protein molecular weight standard marker.

[0112] Step 3: Transfer the protein from the gel to the membrane in the following order: "negative electrode → filter paper → gel → PVDF membrane → filter paper → positive electrode", avoiding air bubbles.

[0113] Step 4: Place the PVDF membrane in the sealing solution and seal it on a shaker at room temperature for 1-2 hours.

[0114] Step 5: Antibody incubation, the specific steps are as follows:

[0115] 6. Place the PVDF membrane in the diluted primary antibody solution and incubate overnight on a shaker at 4°C. Clean the membrane thoroughly with TBST.

[0116] 7. Select a secondary antibody that matches the species of the primary antibody and incubate for 1 hour. Clean the membrane thoroughly with TBST.

[0117] Step 6: Chemiluminescence imaging to detect protein signals.

[0118] like Figure 7 The results showed that both vanillin and L-carnitine-succinate ionic liquid had some inhibitory effect on androgen receptors (AR) in the DHT-constructed HFDPC cell model, but the effect was not ideal. However, the vanillin-loaded L-carnitine-succinate ionic liquid significantly inhibited androgen receptors, restoring them to normal levels. This indicates that vanillin and L-carnitine-succinate ionic liquid can synergistically inhibit androgen receptors and improve androgenetic alopecia.

[0119] Experimental Example 6

[0120] Treatment efficacy for androgenetic alopecia

[0121] 1. Model Building

[0122] Thirty male C57BL / 6 mice (6-8 weeks old, average weight 20 g) were used. Throughout the experiment, the mice were kept at a room temperature of 22-24°C, relative humidity of 60%, and a 12-hour light / dark cycle. Each group of mice was provided with sufficient food and water, and allowed one week to acclimatize to the experimental environment before the experiment. After hair removal with a depilatory cream, a mouse model of androgenetic alopecia was induced by injection of testosterone solution at a dose of 0.1 mL (5 mg / mL). The same dose of testosterone was continuously injected throughout the treatment to maintain androgen levels. Successful model establishment was confirmed by dermoscopy and histopathological examination.

[0123] 2. Evaluation of hair growth in mice

[0124] Experimental mice were randomly divided into a control group, a model group, a vanillin group (Apo), an L-carnitine-succinate ionic liquid group (CSIL), and a vanillin-loaded L-carnitine-succinate ionic liquid group (A@CSIL). The concentrations of vanillin and L-carnitine-succinate ionic liquid in the different groups were 0.5 mg / mL and 1.58 mg / mL, respectively. Healthy controls were injected with saline, and the model group was injected with testosterone solution, with each group receiving 200 μL. Treatment lasted for 21 days. On days 7, 14, and 21, the growth of hair on the backs of the mice was tracked and photographed. Further local observation and recording were performed using a dermoscope, and changes in skin color and hair growth on the backs were recorded in detail.

[0125] like Figure 8 The results showed that both vanillin and L-carnitine-succinate ionic liquids had a certain ameliorative effect on the testosterone-induced androgenetic alopecia model in mice. However, the hair coverage area in the vanillin-loaded L-carnitine-succinate ionic liquid groups was small, and the effect was not ideal. In contrast, the group loaded with vanillin showed significant improvement in androgenetic alopecia, with a significantly increased hair coverage area, returning to the level of normal mice. This demonstrates that vanillin-loaded L-carnitine-succinate ionic liquids can work synergistically to effectively improve androgenetic alopecia.

[0126] In summary, the L-carnitine-succinic acid ionic liquid of this invention not only increases the solubility of vanillin, but also works synergistically with vanillin to improve androgenetic alopecia by promoting angiogenesis, clearing excess reactive oxygen species in the hair follicle microenvironment, inhibiting androgen receptors, and enhancing the activity of dermal papilla cells, which play a key role in androgenetic alopecia.

[0127] While specific embodiments of the invention have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be covered by the appended claims.

Claims

1. An ionic liquid for improving androgenetic alopecia, characterized in that, It is prepared according to the following steps: L-L-carnitine and succinic acid powder were dissolved in ethanol at a ratio of 1 mg: 10 L to 20 L. The reaction was carried out under inert conditions and finally dried under vacuum at 60 °C to 80 °C to obtain an L-L-carnitine-succinic acid ionic liquid with an ion-hydrogen bond network structure. The L-carnitine-succinic acid ionic liquid was dissolved in ethanol and vanillin was heated and stirred to allow vanillin to undergo non-covalent interaction with the ionic-hydrogen bond network structure, thus obtaining the ionic liquid. The molar ratio of L-carnitine to succinic acid is 1:1 to 3; The molar ratio of vanillin ethyl ketone to the L-carnitine-succinic acid ionic liquid is 1:2 to 8; The reaction of L-carnitine and succinic acid under inert conditions was carried out in a nitrogen atmosphere at 50°C to 80°C with stirring at a speed of 200 rpm to 800 rpm for 24 h to 48 h. The heating and stirring temperature is 30–40 °C, the stirring speed is 300–500 rpm, and the stirring time is 1–5 h.

2. The application of the ionic liquid according to claim 1 in the preparation of cosmetics for preventing hair loss and strengthening hair.

3. The application according to claim 2, characterized in that, The ionic liquid is used for androgenetic alopecia.

4. The use of the ionic liquid of claim 1 in the preparation of a drug for promoting hair growth.

5. A cosmetic product for preventing hair loss and strengthening hair, characterized in that, It is formulated by compounding the ionic liquid of claim 1 with acceptable excipients.

6. A drug for promoting hair growth, characterized in that, It is formulated by compounding the ionic liquid of claim 1 with acceptable excipients.

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