Ionic liquid for improving androgenetic alopecia and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to ionic liquid for improving androgenetic alopecia and application of the ionic liquid. The ionic liquid is prepared according to the following steps: carrying out reaction on L-L-carnitine and succinic acid under an inert condition to obtain the L-L-carnitine-succinic acid ionic liquid with an ion-hydrogen bond network structure; and adding acetovanillone into the L-L-carnitine-succinic acid ionic liquid, heating and stirring to obtain the ionic liquid. The ionic liquid can play a role in a microenvironment where hair papilla cells are located, the hair follicle microenvironment is improved, and the activity of the hair papilla cells playing a key role in male hormonal alopecia is improved. In addition, the drug-loaded ionic liquid can effectively inhibit the activity of an androgen receptor, and the problems of hair follicle miniaturization, hair loss and the like caused by the fact that the androgen and the androgen receptor are combined to play a role are solved. Meanwhile, the traditional Chinese medicine composition also has the effects of improving androgenetic alopecia and promoting hair growth.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to an ionic liquid for improving androgenetic alopecia and application thereof. BACKGROUND

[0002] Hair loss is a skin disease characterized by excessive or abnormal hair loss and a decrease in the number of hairs. Androgenetic alopecia (AGA) is one of the most common chronic hair loss types, affecting up to 80% of men and 50% of women, and the incidence often increases with age. Androgenetic alopecia is related to genetics and hormone levels, and genetic predisposition is the underlying cause. The sensitivity of the hair follicles of patients to androgens (especially dihydrotestosterone DHT) is determined by genes, and has a family aggregation. The impact of androgens is not key to the absolute level of androgens (many patients have normal hormone levels), but to the abnormal sensitivity of the hair follicles to DHT. The principle of treating androgenetic alopecia is early intervention, long-term adherence, and combination therapy. The goal is to delay the progression of hair loss and promote hair growth, but it is difficult to regenerate completely necrotic hair follicles.

[0003] The most commonly used way to treat AGA at present is oral finasteride and external minoxidil, but these drugs have certain limitations. For example, minoxidil can cause skin irritation and itching, and finasteride is not suitable for women, and long-term use by men can cause sexual dysfunction. Therefore, there is an urgent need to develop new products that can effectively promote hair regeneration while having good efficacy and biocompatibility. SUMMARY

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

[0005] The specific technical solutions provided by the present application are as follows: In a first aspect, the present application provides an ionic liquid for improving androgenetic alopecia, which is prepared according to the following steps: L-carnitine and succinic acid are reacted under inert conditions to obtain an L-carnitine-succinic acid ionic liquid with an ionic-hydrogen bond network structure through the interaction (Coulomb interaction) between anions and cations and hydrogen bond interaction; Vanillin is added to the L-carnitine-succinic acid ionic liquid, and heated and stirred to allow vanillin to have non-covalent interaction with the ionic-hydrogen bond network structure, thereby obtaining the ionic liquid.

[0006] As a preferred embodiment of the present application, the molar ratio of L-carnitine to succinic acid is 1:1-3.

[0007] As a preferred embodiment of the present application, L-carnitine and succinic acid powder are dissolved in an organic solvent for reaction, and the mass ratio of the sum of the mass of L-carnitine and succinic acid to the mass of the organic solvent is 1 mg: 10-20 L.

[0008] As a preferred embodiment of the present application, the molar ratio of the vanillin and the L-carnitine-succinic acid ionic liquid is 1:2-8.

[0009] As a preferred embodiment of the present application, vanillin and L-carnitine-succinic acid ionic liquid are dissolved in PBS, heated and stirred to obtain L-carnitine-succinic acid ionic liquid loaded with vanillin.

[0010] As a preferred embodiment of the present application, the reaction of the L-carnitine and succinic acid under inert conditions is stirred at a stirring speed of 200 rpm-800 rpm for 24 h-48 h under a nitrogen atmosphere at 50℃-80℃.

[0011] As a preferred embodiment of the present application, the temperature of the heating and stirring is 30-40℃, the stirring speed is 300-500 rpm, and the stirring time is 1 h-5 h.

[0012] As a preferred embodiment of the present application, the reaction product of L-carnitine and succinic acid is vacuum dried at 60-80℃ to obtain L-carnitine-succinic acid ionic liquid with an ionic-hydrogen bond network structure.

[0013] In a second aspect of the present application, the ionic liquid is provided for use in the preparation of a cosmetic for preventing hair loss.

[0014] The L-carnitine-succinic acid ionic liquid loaded with vanillin (Apo) has the effects of antioxidant, androgen receptor inhibition, and activation of the β-catenin / Wnt3a pathway.

[0015] As a preferred embodiment of the present application, the ionic liquid is used for androgenic alopecia.

[0016] In a third aspect of the present application, the ionic liquid is provided for use in the preparation of a drug for promoting hair growth.

[0017] In a fourth aspect, the present application provides a cosmetic product for preventing hair loss, which is prepared by compounding the ionic liquid with acceptable adjuvants. The acceptable adjuvants include, but are not limited to, surfactants (such as sodium lauroyl glutamate, sodium methyl cocoyl taurate, sodium laureth sulfate, sodium lauryl sulfate, cocamidopropyl betaine, cocamide MEA, alkyl polyglycoside), conditioning agents (such as polyquaternium-10, polyquaternium-7, dimethicone, cyclomethicone, natural oils and esters), thickening agents (such as xanthan gum, carbomer, hydroxyethyl cellulose), pH adjusters (citric acid, lactic acid, sodium hydroxide, triethanolamine, etc.), preservatives (such as phenoxyethanol, caprylyl hydroxamic acid, p-hydroxyacetophenone, ethylhexylglycerin, sodium benzoate, potassium sorbate), penetration enhancers (such as azone, nicotinic acid ethyl ester), antioxidants (such as vitamin E, disodium EDTA), and the like.

[0018] In a fifth aspect, the present application provides a medicament for promoting hair growth, which is prepared by compounding the ionic liquid with acceptable adjuvants. The acceptable adjuvants 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, and the like.

[0019] Compared with the prior art, the present application has the following advantages: The present application provides an ionic liquid for improving androgenetic alopecia, in which vanillylacetone (Apo), L-levocarnitine (LC) and succinic acid (SA) respectively assume three types of functions of drug loading activity, ionic liquid cation / anion pairing and hair follicle microenvironment regulation in the system, thereby producing synergies at the following levels: Firstly, carrier level synergy (dissolution and delivery): the ionic liquid (CSIL) composed of LC / SA interacts with Apo through ionic-hydrogen bond network to improve the dispersion and availability of Apo in aqueous phase / PBS, overcome the hydrophobicity limitation and ensure the effective exposure of Apo in the microenvironment of hair follicle dermal papilla cells (HFDPC).

[0020] Secondly, microenvironment level synergy (antioxidation → metabolic homeostasis): Apo reduces the ROS content in the microenvironment by inhibiting NADPH oxidase; LC and SA in the CSIL component are respectively involved in fatty acid mitochondrial transport and tricarboxylic acid cycle to promote cellular energy metabolic homeostasis, and the combination of the two with Apo realizes the bidirectional regulation of ROS reduction and cell metabolism promotion, thereby improving the hair follicle microenvironment.

[0021] Thirdly, target / pathway level synergy (AR and Wnt pathway): In DHT-induced model, Apo combined with CSIL showed stronger inhibition on androgen receptor (AR) activity than single component, accompanied by activation of β-catenin / Wnt3a signaling pathway, thus promoting hair papilla cell migration and angiogenesis.

[0022] Fourthly, integrated effect: The above multi-level synergy ultimately manifested as a significant increase in hair coverage in a mouse AGA model, which was superior to Apo or CSIL alone. See Figures 4-8 Results and controls.

[0023] The drug-loaded ionic liquid provided by the application can not only effectively improve the water solubility of vanillin ketone, but also effectively inhibit the androgen receptor, avoid the conversion of testosterone into DHT catalyzed by 5α-reductase, and act on the hair follicle after binding with the androgen receptor to produce rapid regression, miniaturization, hair loss and other problems. At the same time, the drug-loaded ionic liquid can also inhibit the production of active oxygen in the skin hair follicle to achieve the purpose of preventing hair loss and promoting hair growth. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is an actual picture of the prepared L-leucine carnitine-succinic acid ionic liquid.

[0025] Figure 2 It is a TEM picture of the prepared L-leucine carnitine-succinic acid ionic liquid.

[0026] Figure 3 It is the blood compatibility of different samples.

[0027] Figure 4 It is the clearance rate of different samples on hydroxyl radicals.

[0028] Figure 5 It is the effect of different concentration samples on the migration rate of hair papilla cells. A, cell migration change, B, cell migration rate statistical chart.

[0029] Figure 6 It is the effect of different concentration samples on the angiogenesis rate of human umbilical vein endothelial cells. A, total blood vessel length, B, blood vessel connection point, C, tube formation image of HUVEC cells after different treatments.

[0030] Figure 7 It is the effect of different samples on AR.

[0031] Figure 8 It is a schematic diagram of the hair coverage during the treatment of mice. DETAILED DESCRIPTION

[0032] In order to make the above objectives, characteristics and advantages of the present application more apparent, comprehensible and easier to be understood, the specific embodiments of the present application will be described in detail below with reference to the examples.

[0033] The present application provides a vanillyl ketone-loaded L-carnitine-succinic acid ionic liquid, which is prepared according to the following steps: Step one: L-carnitine and succinic acid powder are dissolved in an organic solvent, L-carnitine and succinic acid are added according to 1:1-3, stirred under nitrogen atmosphere at 50-80 ℃, 200-800 rpm for 24-48 h, and vacuum dried at 60-80 ℃ to obtain L-carnitine-succinic acid ionic liquid.

[0034] Step two: Apo and L-carnitine-succinic acid ionic liquid are dissolved in ethanol, and stirred at 30-40 ℃, 300-500 rpm to obtain a vanillyl ketone-loaded L-carnitine-succinic acid ionic liquid.

[0035] The vanillyl ketone is a natural product with wide biological activity and is a natural NADPH oxidase inhibitor. However, the physical and chemical properties of vanillyl ketone show that vanillyl ketone is insoluble in water at room temperature, the molecular structure is relatively hydrophobic, and the poor water solubility of vanillyl ketone limits its application in improving androgenic alopecia. In addition, the L-carnitine-succinic acid ionic liquid can effectively improve the solubility of vanillyl ketone. In addition, the L-carnitine-succinic acid ionic liquid also has the effect of promoting hair growth. Therefore, the L-carnitine-succinic acid ionic liquid prepared by the present application can not only act as a carrier for vanillyl ketone, but also can play a synergistic effect with vanillyl ketone to improve androgenic alopecia.

[0036] The ionic liquid can play a role in the microenvironment of the hair papilla cells, improve the microenvironment of the hair follicle, and improve the activity of the hair papilla cells which play a key role in androgenic alopecia. Moreover, the drug-loaded ionic liquid can effectively inhibit the activity of androgen receptors, avoid the combination of androgens and androgen receptors to play a role, and make the hair follicle quickly enter the involutional stage, leading to problems such as miniaturization of the hair follicle and hair loss. At the same time, it can also inhibit the production of ROS in the skin hair follicle, promote angiogenesis, activate the β-catenin / Wnt3a pathway, and achieve the purpose of improving androgenic alopecia and promoting hair growth.

[0037] Example 1 A vanillyl ketone-loaded L-carnitine-succinic acid ionic liquid is prepared according to the following steps: ​Step one: L-carnitine and succinic acid powder were dissolved in ethanol, the molar ratio of L-carnitine and succinic acid was 1:3, the solid-liquid ratio was 1 mg:10 L, heated at 50 ℃ under nitrogen atmosphere, 500 rpm stirring for 24 h, and finally dried under vacuum at 60 ℃ to remove ethanol, obtaining a light yellow sticky L-carnitine succinic acid ionic liquid.

[0038] Step two: vanillin and L-carnitine-succinic acid ionic liquid were dissolved in ethanol, the molar ratio of vanillin and L-carnitine-succinic acid ionic liquid was 1:4, heated at 35 ℃, 500 rpm stirring for 4 h, obtaining vanillin loaded L-carnitine-succinic acid ionic liquid.

[0039] Example 2 A vanillin loaded L-carnitine-succinic acid ionic liquid was prepared according to the following steps: Step one: L-carnitine and succinic acid powder were dissolved in ethanol, the molar ratio of L-carnitine and succinic acid was 1:3, the solid-liquid ratio was 1 mg:20 L, heated at 60 ℃ under nitrogen atmosphere, 500 rpm stirring for 36 h, and finally dried under vacuum at 70 ℃ to remove ethanol, obtaining a light yellow sticky L-carnitine-succinic acid ionic liquid.

[0040] Step two: vanillin and L-carnitine-succinic acid ionic liquid were dissolved in ethanol, the molar ratio of vanillin and L-carnitine-succinic acid ionic liquid was 1:2, heated at 40 ℃, 300 rpm stirring for 5 h, obtaining vanillin loaded L-carnitine-succinic acid ionic liquid.

[0041] Example 3 A vanillin loaded L-carnitine-succinic acid ionic liquid was prepared according to the following steps: Step one: L-carnitine and succinic acid powder were dissolved in ethanol, the molar ratio of L-carnitine and succinic acid was 1:2, the solid-liquid ratio was 1 mg:10 L, heated at 50 ℃ under nitrogen atmosphere, 800 rpm stirring for 24 h, and finally dried at 50 ℃ under vacuum, obtaining a light yellow sticky L-carnitine-succinic acid ionic liquid.

[0042] Step two: vanillin and L-carnitine-succinic acid ionic liquid were dissolved in ethanol, the molar ratio of vanillin and L-carnitine-succinic acid ionic liquid was 1:8, heated at 30 ℃, 500 rpm stirring for 1 h, obtaining vanillin loaded L-carnitine-succinic acid ionic liquid.

[0043] Example 4 A vanillin-loaded L-carnitine-succinate ionic liquid is prepared according to the following steps: Step one: L-carnitine and succinic acid powders are dissolved in ethanol, L-carnitine and succinic acid are fed according to a molar ratio of 1:2, the liquid-to-solid ratio is 1 mg:10 L, heated at 70 °C under a nitrogen atmosphere, stirred at 800 rpm for 48 h, and finally dried under vacuum at 70 °C to obtain a yellowish viscous L-carnitine-succinate ionic liquid.

[0044] Step two: vanillin and the L-carnitine-succinate ionic liquid are dissolved in ethanol, the molar ratio of vanillin to the L-carnitine-succinate ionic liquid is 1:4, heated at 30 °C, and stirred at 500 rpm for 2 h to obtain a vanillin-loaded L-carnitine-succinate ionic liquid.

[0045] Example 5 A vanillin-loaded L-carnitine-succinate ionic liquid is prepared according to the following steps: Step one: L-carnitine and succinic acid powders are dissolved in ethanol, L-carnitine and succinic acid are fed according to a molar ratio of 1:2, the liquid-to-solid ratio is 1 mg:10 L, heated at 70 °C under a nitrogen atmosphere, stirred at 800 rpm for 48 h, and finally dried under vacuum at 70 °C to obtain a yellowish viscous L-carnitine-succinate ionic liquid.

[0046] Step two: vanillin and the L-carnitine-succinate ionic liquid are dissolved in ethanol, the molar ratio of vanillin to the L-carnitine-succinate ionic liquid is 1:4, heated at 30 °C, and stirred at 500 rpm for 2 h to obtain a vanillin-loaded L-carnitine-succinate ionic liquid.

[0047] Comparative Example 1 L-carnitine and succinic acid powders are dissolved in ethanol, L-carnitine and succinic acid are fed according to a molar ratio of 3:1, the liquid-to-solid ratio is 1 mg:10 L, heated at 30 °C under a nitrogen atmosphere, stirred at 500 rpm for 24 h, and finally dried under vacuum at 70 °C to obtain a white solid powder.

[0048] It is found that when the reaction temperature in step one is lower than 40 °C, the ionic liquid cannot be formed. According to Comparative Example 1, the ionic liquid cannot be formed when the molar ratio is 3:1, and a white solid powder is finally obtained.

[0049] Comparative Example 2 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.

[0050] 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.

[0051] 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.

[0052] Experimental Example 1 Biocompatibility testing 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:

[0053] Where As represents the absorbance of different samples, Ap represents the absorbance of deionized water, and An represents the absorbance of physiological saline.

[0054] Depend on Figure 3 It is known that the L-L-carnitine succinate ionic liquid loaded with vanillin has good biocompatibility.

[0055] Experimental Example 2 Hydroxyl radical (·OH) scavenging rate The FeSO4, H2O2 solution was equipped with a concentration of 2 mM, and the concentration ratio of the FeSO4, H2O2 solution was 1:1. The samples were divided into a blank control group, a vanillin acetone group (Apo), an L-carnitine-succinic acid ionic liquid group (CSIL), and a vanillin acetone-loaded L-carnitine-succinic acid ionic liquid group (A@CSIL), wherein the concentrations of vanillin acetone and L-carnitine-succinic acid ionic liquid in different groups were 0.5 mg / mL and 1.58 mg / mL, respectively. The samples in different groups were added to a 37 ℃ reaction for 2 h, and the reaction liquid was taken for the next reaction with 3,3',5',5-tetramethylbenzidine at a volume ratio of 1:1. After 15 min of reaction, the solutions in each group were transferred to a 96-well plate, and the absorbance at 650 nm was measured. The hydroxyl radical scavenging rate was calculated according to the following formula:

[0056] wherein As is the absorbance corresponding to different samples, An is the absorbance corresponding to deionized water, and Ap is the absorbance corresponding to the blank control group.

[0057] As shown in Figure 4 The vanillin acetone and L-carnitine-succinic acid ionic liquid had hydroxyl radical scavenging rates of 53.86% and 33.57%, respectively, and the scavenging effect was not ideal. The L-carnitine-succinic acid ionic liquid loaded with vanillin acetone had a hydroxyl radical scavenging rate of 86.18%, indicating that vanillin acetone and L-carnitine-succinic acid ionic liquid could synergistically scavenge excess active oxygen in the hair follicle microenvironment.

[0058] Experimental Example 3 Hair follicle cell migration rate detection experiment Cell migration rate: the cell scratch test was used to determine the cell migration ability, and the experiment was divided into four links: culture plate marking, cell inoculation, cell scratch, and observation of cell healing. The specific operation steps are as follows: a. Culture plate marking: invert the 6-well plate on the desktop, and evenly mark horizontal lines on the bottom of the well plate with a ruler, about every 0.5-1 cm, crossing the well. At least 3 lines per well.

[0059] b. Cell inoculation: inoculate about 5×10 5 cells in a total of 2 mL into the 6-well culture plate, and culture in a 37 ℃ CO2 incubator until the cells adhere and cover the bottom of the well plate.

[0060] c. Cell scratch: Take out the culture plate, observe the cell state, and place the steel ruler on the six-well plate after sterilization. Use a sterile 200 μL syringe to draw a line in the center of the cells, perpendicular to the horizontal line drawn in step a. Keep the syringe as straight as possible while drawing the line, and do not tilt it. Then discard the original culture medium, wash the cells with PBS three times to remove the cells removed by scratching. Place the well plate under a microscope and take a photo at 0 h.

[0061] d. The experiment is divided into five groups, namely the blank group, the DHT group, the vanillin group (Apo), the L-carnitine-succinate ionic liquid group (CSIL), and the L-carnitine-succinate ionic liquid loaded with vanillin group (A@CSIL). The blank group is added with serum-free medium, and the other groups are added with samples diluted with serum-free medium. The concentrations of vanillin and L-carnitine-succinate ionic liquid in the samples are 332 ng / mL and 1 μg / mL, respectively. Place the culture plate in a 37 ℃, 5% CO2 incubator and continue to culture for 12 h. Take a photo under a microscope at 12 h (keep the same position as at 0 h) to observe the repair of the cell scratch. Analyze the area of the scratch region using Image J software, and calculate the cell migration rate by the formula:

[0062] where A0 and At are the initial scratch area and the scratch area after 0 h and 12 h of culture, respectively.

[0063] Cell culture and passage: Take out the frozen tube of hair follicle cells stored in liquid nitrogen, and place it in a 37 ℃ water bath for shaking. After the frozen solution is completely melted, add it to the primary fibroblast cell basic medium (containing 10% fetal bovine serum and 1×10 5 U / L penicillin, 100 mg / L streptomycin) T25 cell culture bottle, shake it evenly with the cross method, and then place the culture bottle in a 37 ℃, 5% CO2, and humidity-saturated cell culture incubator. Observe the cell adhesion after 4 h and continue to culture.

[0064] Cell density exceeds 80%: aspirate the cell culture solution, gently blow and rinse twice with PBS, add trypsin containing 0.25% EDTA, and digest in the incubator for 2 min. Stop the cell digestion with fresh primary fibroblast cell basic medium. Centrifuge the cell suspension at 1500 rpm for 5 min. Blow the cell pellet with fresh primary fibroblast cell basic medium, and determine the number of passage bottles according to the cell number, state, and experimental requirements. Place the culture bottle in the cell culture incubator for culture.

[0065] For example, Figure 5It is shown that both vanillin and L-carnitine-succinate ionic liquid have certain effects on the cell migration of the HFDPC cell model constructed by DHT, but the L-carnitine-succinate ionic liquid loaded with vanillin has more obvious effect on promoting cell migration. Among them, the cell migration rates of the normal group and the cell model group constructed by DHT are 44.73% and 20.22% respectively; the cell migration rates of the vanillin group and the L-carnitine-succinate ionic liquid group are 34.92% and 45.34% respectively, and the cell migration rate of the L-carnitine-succinate ionic liquid loaded with vanillin group is 50%. The results show that both vanillin and L-carnitine-succinate ionic liquid can improve the inhibition of DHT on cell migration, so that the cell migration rate returns to the normal level, and synergistically promotes cell migration.

[0066] Experimental Example 4 Angiogenesis rate detection experiment Human umbilical vein endothelial cell (HUVEC) angiogenesis experiment: after the HUVEC cells were cultured for 24 h and adhered, the culture medium was aspirated, and the plate bottom was washed with PBS for 2 times. 2 mL of freshly prepared drug was added to each well, and a blank group and sample groups with different mass concentrations were set, and the culture was continued for 48 h.

[0067] Cell culture and subculture: the HUVEC cell cryopreservation tube stored in liquid nitrogen was taken out and subjected to 37°C water bath oscillation, and after the cryopreservation liquid was completely melted, it was quickly added to a T25 cell culture bottle containing DMEM culture medium (containing 10% fetal bovine serum and 1×10 5 U / L penicillin, 100 mg / L streptomycin) by a cross method, and then the culture bottle was placed in a cell culture box at 37°C, 5% CO2 and saturated humidity for culture. After 4 h of observation of cell adhesion, the liquid was replaced and the culture was continued.

[0068] Cell density over 80% cell subculture: the cell culture liquid was aspirated, and PBS was gently blown and washed twice. 0.25% trypsin containing EDTA was added, and the culture was digested in the incubator for 3 min. The cell digestion was terminated by using fresh DMEM complete culture medium. The cell suspension was centrifuged at 1500 rpm for 5 min. Fresh DMEM complete culture medium was added to blow the cell precipitate, and the number of subculture bottles was determined according to the number of cells, state and experimental requirements. The culture bottle was placed in the cell culture box for culture.

[0069] As Figure 6The results showed that the normal group had 115 branch points and the total blood vessel length was 11524 μm; the DHT constructed cell model group had 45 branch points and the total blood vessel length was 7253 μm; the vanillin group had 61 branch points and the total blood vessel length was 9000 μm; the L-carnitine-succinate ionic liquid group had 81 branch points and the total blood vessel length was 10215 μm; and the L-carnitine-succinate ionic liquid loaded with vanillin group had 94 branch points and the total blood vessel length was 10816 μm, and the degree of angiogenesis returned to the normal level. The results showed that vanillin and L-carnitine-succinate ionic liquid had a synergistic effect on promoting angiogenesis of the DHT constructed HFDPC cell model.

[0070] Experimental Example 5 Western blotting test (WB) detects the activation pathway The expression of key proteins in the pathway is detected by WB test to determine whether the drug regulates the target pathway. The specific operation steps are as follows: Step one: protein extraction, the specific operation steps are as follows: 0. Take different drug treated 6 hole plate, discard the culture medium, wash the cells with pre-cooled PBS for 2-3 times; 1. Add 150-200 μL of RIPA lysis solution containing protease / phosphatase inhibitor to each well, lyse for 30 min on ice (during which use a pipette to repeatedly blow to ensure that the cells are fully lysed); 2. Centrifuge at 12000 rpm for 15 min at 4℃, and aspirate the supernatant (i.e. protein sample) into a new centrifuge tube, and discard the precipitate (cell fragments); 3. Detect the protein concentration by BCA method or Bradford method (avoid differences in sample amount), dilute all samples to the same concentration (such as 2 μg / μL) according to the quantitative results, then add 5×SDS loading buffer (final concentration 1×), and boil at 95℃ for 5 min to denature the protein, and store at -20℃ for standby use (short term 1 week, long term -80℃).

[0071] Step two: SDS-PAGE gel electrophoresis 4. Gel preparation: configure the separation gel according to the protein molecular weight, pour into the electrophoresis tank, and insert the comb, and solidify at room temperature for 30 min (avoid bubbles, otherwise affect electrophoresis); 5. Sample loading and electrophoresis: add 1×SDS electrophoresis buffer to the electrophoresis tank, remove the comb, and load 20-50 μg of protein sample per well, and load protein molecular weight marker at the same time.

[0072] Step three: Transfer the protein on the gel to the membrane, in the order of "negative electrode→filter paper→gel→PVDF membrane→filter paper→positive electrode", avoiding bubbles.

[0073] Step four: Put the PVDF membrane into the blocking solution and block it in the shaker at room temperature for 1-2 hours.

[0074] Step five: Incubate the antibody, and the specific operation steps are as follows: 6. Put the PVDF membrane into the diluted primary antibody solution and incubate it in the shaker at 4℃ overnight. Wash the membrane with TBST. 7. Incubate the secondary antibody matched with the species of the primary antibody for 1 hour, and wash the membrane with TBST. Step six: Chemiluminescence development, detect the protein signal.

[0075] As Figure 7 It is shown that both vanillin and L-carnitine-succinate ionic liquid have a certain inhibitory effect on androgen receptor (AR) in the HFDPC cell model constructed by DHT, but the effect is not ideal. The L-carnitine-succinate ionic liquid loaded with vanillin has a significant inhibitory effect on androgen receptor, and the androgen receptor returns to the normal level. It is shown that vanillin and L-carnitine-succinate ionic liquid can synergistically inhibit androgen receptor to improve androgenic alopecia.

[0076] Experimental Example 6 Therapeutic effect of androgenic alopecia 1. Model construction Prepare 30 male C57BL / 6 mice (6-8 weeks old, average weight 20 g). The whole experimental period is kept at room temperature of 22-24℃, relative humidity of 60%, 12 h light / dark cycle, and ensure that each group of experimental mice has enough food and water, and allow the mice to adapt to the experimental environment for one week before the experiment. After depilation with depilatory cream, inject testosterone solution to induce androgenic alopecia mouse model. The dose is 0.1 mL, 5 mg / mL. Continue to inject the same dose of testosterone during the whole treatment process to maintain the level of androgen in the body. Determine the success of the model by skin examination and histopathological section.

[0077] 2. Evaluation of mouse hair growth The experimental mice are divided into a control group, a model group, a vanillin ketone group (Apo), an L-sarcosine succinate ionic liquid group (CSIL), and a vanillin ketone-loaded L-sarcosine succinate ionic liquid group (A@CSIL). The concentrations of vanillin ketone and L-sarcosine succinate ionic liquid in different groups are 0.5 mg / mL and 1.58 mg / mL, respectively. The healthy control group is injected with normal saline, and the model group is injected with a testosterone solution, with an injection dose of 200 μL for each group. The treatment lasts for 21 days. On the 7th day, the 14th day, and the 21st day, the growth of the back hair of the mice is tracked and photographed, further local observation is recorded using a dermatoscope, and the changes in the color of the back skin and the hair growth condition are recorded in detail.

[0078] As Figure 8 It is shown that both vanillin ketone and L-sarcosine succinate ionic liquid have a certain improvement effect on the testosterone-induced mouse androgenic alopecia model, but the hair coverage area of the vanillin ketone and L-sarcosine succinate ionic liquid groups alone is small, and the effect is not ideal. The androgenic alopecia of the mice in the vanillin ketone-loaded L-sarcosine succinate ionic liquid group is obviously improved, the hair coverage area is obviously increased, and is restored to the level of normal mice. It can be proved that the vanillin ketone-loaded L-sarcosine succinate ionic liquid can synergistically play a role and effectively improve androgenic alopecia.

[0079] In summary, the L-sarcosine succinate ionic liquid in the present application not only increases the solubility of vanillin ketone, but also can synergistically play a role with vanillin ketone in promoting angiogenesis, removing excess active oxygen in the microenvironment of hair follicles, inhibiting androgen receptors, and improving the activity of hair papilla cells which play a key role in androgenic alopecia, and the like to improve androgenic alopecia.

[0080] Although the specific embodiments of the present application have been illustrated and described, it is obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the present application. Therefore, it is intended to cover all such changes and modifications which fall within the scope of the present application in the appended claims.

Claims

1. An ionic liquid for improving androgenetic alopecia, characterized by comprising It is prepared according to the following steps: L-carnitine and succinic acid are reacted under inert conditions to obtain L-carnitine-succinic acid ionic liquid with ion-hydrogen bond network structure; Vanillin ketone is added to the L-carnitine-succinic acid ionic liquid, and heated and stirred to make vanillin ketone and the ion-hydrogen bond network structure have non-covalent interaction, thereby obtaining the ionic liquid.

2. The ionic liquid of claim 1, wherein, The molar ratio of the L-carnitine and succinic acid is 1:1-3.

3. The ionic liquid of claim 1, wherein, The molar ratio of the vanillin ketone and the L-carnitine-succinic acid ionic liquid is 1:2-8.

4. The ionic liquid of claim 1, wherein, The reaction of the L-carnitine and succinic acid under inert conditions is carried out under nitrogen environment, at 50-80 DEG C, with stirring speed of 200 rpm-800 rpm, and stirring for 24-48 h.

5. The ionic liquid of claim 1, wherein, The temperature of the heating and stirring is 30-40 DEG C, the stirring speed is 300-500 rpm, and the stirring time is 1-5 h.

6. Use of the ionic liquid of any one of claims 1-5 in the preparation of a cosmetic for preventing hair loss.

7. Use according to claim 6, characterized in that, The ionic liquid is used for androgenic alopecia.

8. Use of the ionic liquid of any one of claims 1-5 in the preparation of a drug for promoting hair growth.

9. A cosmetic for preventing hair drop, characterized by, It is compounded from the ionic liquid of any one of claims 1-5 and acceptable adjuvants.

10. A medicament for promoting hair growth, characterized by, It is compounded from the ionic liquid of any one of claims 1-5 and acceptable adjuvants.

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