Hair growth liniment and preparation method thereof

Abstract

The invention discloses a hair-growing liniment and a preparation method thereof, and belongs to the technical field of pharmacy. The liniment comprises the following components in percentage by weight: 1-10% of minoxidil microspheres, 5-20% of a film-forming agent, 0.5-5% of a polypeptide compound, 5-30% of a liquid auxiliary material and the balance of deionized water. Wherein the minoxidil microspheres are of a three-layer structure composed of minoxidil in the inner layer, nano enzyme (cerium dioxide) in the middle layer and lactic acid-glycolic acid ester copolymer or chitosan in the outer layer, and controlled release and targeted delivery are achieved; the polypeptide compound is a compound of oldenlandia diffusa polypeptide and a copper curcumin derivative, and has the effect of synergistically repairing a hair follicle microenvironment. Tests prove that the liniment can remarkably prolong the release time of minoxidil, increase the retention volume of minoxidil in hair follicle parts and reduce absorption of the whole body, shows a hair growth promoting effect superior to that of a traditional preparation and a single-layer microsphere preparation in an animal model, and has good stability and use comfort.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, specifically relating to a hair growth liniment and its preparation method. Background Technology

[0002] Hair loss has become a common skin and hair health problem worldwide. Its causes include a variety of factors such as genetics, endocrine disorders, excessive mental stress, damage to the hair follicle microenvironment, and poor blood circulation in the scalp. It not only affects appearance but also easily causes psychological stress for patients. Therefore, the research and optimization of topical hair growth preparations has become a research hotspot in the pharmaceutical and daily chemical fields.

[0003] Minoxidil, as a clinically recognized effective active ingredient for promoting hair growth, is widely used in various hair growth products. However, traditional topical minoxidil formulations have significant technical drawbacks: First, minoxidil itself has poor water solubility, low transdermal absorption efficiency, and is easily lost quickly on the scalp surface, making it difficult to continuously act on the hair follicle target, resulting in low bioavailability. Second, the hair growth mechanism of single minoxidil formulations is relatively simple, with limited effects on repairing the hair follicle microenvironment and inhibiting inflammation, and poor improvement effects on multifactorial hair loss. Third, traditional formulations lack controlled-release effects, requiring frequent application, and some formulations are prone to causing problems such as oily scalp and dust adhesion, reducing patient compliance.

[0004] To address the delivery challenges of minoxidil, existing technologies have attempted controlled release through microsphere encapsulation. However, conventional minoxidil microspheres are mostly monolayer structures, enabling only simple, slow release and failing to simultaneously promote transdermal absorption and protect hair follicles. Furthermore, some microsphere carrier materials lack biocompatibility, potentially irritating the scalp. Simultaneously, existing hair growth formulations often feature relatively simple active ingredient combinations, primarily based on minoxidil, lacking active components that synergistically repair hair follicles and improve the scalp microenvironment. While peptides and plant extracts, which promote hair follicle activation, are used in limited quantities, their poor compatibility with minoxidil and tendency to inactivate limit their synergistic effects.

[0005] Furthermore, the film-forming system design of existing hair growth lotions is flawed. Some film-forming agents have poor breathability after film formation, easily clogging hair follicles and exacerbating hair loss; others have weak film-forming properties, failing to effectively fix active ingredients and causing them to be quickly lost. At the same time, the selection of excipients for topical formulations is also limited, making it difficult to simultaneously meet the multiple needs of moisturizing, soothing the scalp, and promoting the absorption of active ingredients. Moreover, some excipients are incompatible with active ingredients, affecting the stability of the formulation.

[0006] In summary, there is an urgent need for a novel hair growth topical preparation that can address the issues of poor transdermal absorption and low bioavailability of traditional minoxidil formulations through rational component compatibility and structural design. This would enable controlled release and targeted delivery of active ingredients, while simultaneously repairing the hair follicle microenvironment and activating dormant hair follicles through the synergistic effect of multiple active ingredients. Combined with a suitable film-forming system and excipients, this preparation would balance stability, user comfort, and hair growth promotion effects, meeting the clinical and market demands for a highly effective, safe, and convenient topical hair growth preparation. Summary of the Invention

[0007] To address the shortcomings of traditional minoxidil-based hair growth lotions, such as low transdermal absorption efficiency, poor bioavailability, easy loss of active ingredients, and limited efficacy, this application provides a hair growth lotion and its preparation method. It utilizes three-layered minoxidil microspheres to achieve controlled release and targeted delivery of active ingredients. A multi-component synergistic hair growth-promoting effect is achieved by combining a Hedyotis diffusa polypeptide-copper curcumin derivative polypeptide complex. Furthermore, a suitable film-forming agent and liquid excipients are used to construct a stable lotion system, thus solving the technical problems of low utilization rate of active ingredients and limited repair effect on the hair follicle microenvironment in traditional formulations.

[0008] This application provides a hair growth liniment, which comprises, by weight percentage: 1-10% minoxidil microspheres, 5-20% film-forming agent, 0.5-5% polypeptide complex, 5-30% liquid excipients, and the balance being deionized water.

[0009] Preferably, the minoxidil microspheres have a three-layer structure, with an inner layer of minoxidil, a middle layer of nanozyme, and an outer layer of any one of lactic acid-glycolic acid copolymer or chitosan.

[0010] Preferably, the polypeptide complex is a complex of Hedyotis diffusa polypeptide and copper curcumin derivative.

[0011] Preferably, the nanozyme is cerium dioxide.

[0012] Preferably, the mass ratio of the Hedyotis diffusa polypeptide to the copper curcumin derivative is 1:1 to 5.

[0013] Preferably, the film-forming agent is either hydroxypropyl methylcellulose or carbomer.

[0014] Preferably, the liquid excipient is any one of glycerol, butylene glycol, or menthol.

[0015] This application also provides a method for preparing a hair growth liniment, the method comprising the following steps:

[0016] (1) Add the film-forming agent to deionized water, allow it to swell fully and stir until homogeneous to obtain the film-forming solution;

[0017] (2) Add the liquid excipients to the film-forming solution and stir to mix evenly to obtain the intermediate product;

[0018] (3) Add minoxidil microspheres and polypeptide complex to the intermediate in sequence, stir and disperse evenly, add deionized water, and continue stirring until the system is homogeneous and stable to obtain hair growth liniment.

[0019] Preferably, the method for preparing the minoxidil microspheres includes the following steps:

[0020] (1) Minoxidil and polymer materials are dissolved in an organic solvent, cerium dioxide nanozyme is added and dispersed evenly to obtain an oil phase;

[0021] (2) The oil phase is added to the aqueous phase containing the emulsifier and emulsified to form an emulsion;

[0022] (3) Stirring to evaporate the organic solvent and solidify the microspheres;

[0023] (4) Centrifuge, wash and dry to obtain a three-layer structure of minoxidil microspheres with an inner layer of minoxidil, a middle layer of cerium dioxide nanozyme and an outer layer of lactic acid-hydroxyacetic acid copolymer or chitosan.

[0024] Preferably, the method for preparing the polypeptide complex includes the following steps:

[0025] (1) Dissolve the white flower snake tongue grass polypeptide in deionized water to obtain a polypeptide solution;

[0026] (2) Dissolve the copper curcumin derivative in an organic solvent to obtain a copper curcumin derivative solution;

[0027] (3) Add the copper curcumin derivative solution dropwise to the polypeptide solution, stir, purify and dry to obtain the polypeptide complex.

[0028] The beneficial effects of the embodiments in this application are as follows:

[0029] (1) The three-layer structure of minoxidil microspheres designed in this application: the inner layer of minoxidil is the core of the drug efficacy for the treatment of androgenetic alopecia, which acts directly on the hair follicle and inhibits dihydrotestosterone; the middle layer is cerium dioxide, which has antioxidant activity and can remove excess reactive oxygen species in the microenvironment of the hair follicle and alleviate the damage of oxidative stress to the hair follicle; the outer layer of lactic acid-hydroxyacetic acid copolymer or chitosan is a barrier layer, which prevents the rapid loss of minoxidil by regulating the polymer degradation rate, and achieves slow and long-lasting drug release, fundamentally solving the problem that traditional formulations need to be applied multiple times a day. In vitro release tests showed that the cumulative release rate of the topical agent of this application was 67.5%~72.6% over 24 hours, significantly lower than 89.2% for monolayer microsphere formulations and 95.6% for commercially available formulations. The release curve was smooth with no sudden release, effectively prolonging the duration of drug action. Transdermal absorption tests showed that the drug retention in the hair follicle-rich dermal layer of this invention reached 12.3~13.5 μg / cm², which is 1.5~1.7 times that of commercially available formulations. Meanwhile, the amount of drug in the transdermal receiving fluid was significantly lower than that in the control group, achieving targeted enrichment of the drug in the hair follicles and reducing potential side effects caused by systemic absorption.

[0030] (2) The peptide complex was constructed by the peptide of Hedyotis diffusa and the copper curcumin derivative. The peptide of Hedyotis diffusa is rich in active amino acid sequences, which can activate the proliferation of dermal papilla cells and upregulate the expression of hair growth-related growth factors. The copper ions in the copper curcumin derivative participate in the activation of hair follicle metabolic enzymes as trace elements. Curcumin has anti-inflammatory and antioxidant effects, which can improve the microenvironment of hair follicles. The peptide complex activates dermal papilla cells and can promote hair follicle regeneration. Cerium dioxide and curcumin together scavenge reactive oxygen species and improve the microenvironment of animal hair growth. In the experiment, the hair follicles on the back of mice in the topical application group entered the growth phase 7-10 days after administration, which was faster than the single-layer microsphere group (9-12 days), the group without peptide complex (10-13 days) and the commercial group (12-15 days). On the 28th day, the hair completely covered the hair loss area, while the control group was still pink with no new hair. This proves that the three-layer microspheres and the peptide complex have a significant synergistic effect in antagonizing androgenic hair follicle damage.

[0031] (3) Hydroxypropyl methylcellulose or carbomer are used as film-forming agents to form a flexible film with good breathability and without clogging hair follicles on the scalp surface. This film can effectively fix minoxidil microspheres and polypeptide complexes to the site of action and prevent the loss of active ingredients due to friction and sweating. Glycerin, butylene glycol or menthol are used as liquid excipients to provide a moisturizing, soothing and cooling experience and promote the transdermal absorption of active ingredients by adjusting the osmotic pressure of the system.

[0032] This solves the shortcomings of existing hair growth lotions, such as low transdermal absorption efficiency, poor bioavailability, easy loss of active ingredients, and single function. Attached Figure Description

[0033] Figure 1 This is a graph showing the amount of minoxidil in the transdermal receiving fluid of the test group in Experiment Example 2 of this application.

[0034] Figure 2 The diagram shows the hair growth of Comparative Example 3 and Example 1 in Test Example 3 of this application; where A is Comparative Example 3 and B is Example 1. Detailed Implementation

[0035] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0036] In the following examples, curcumin (purity ≥99%) was purchased from Hubei Tuoyuan Fine Chemical Co., Ltd.; minoxidil (purity ≥99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; nano-cerium dioxide was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; lactic acid-glycolic acid copolymer was purchased from Zhejiang Hisun Biomaterials Co., Ltd.; chitosan was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; Hedyotis diffusa was purchased from Jiangxi Kangzhikang Traditional Chinese Medicine Technology Co., Ltd.; and other reagents and equipment not specified were conventional and commercially available.

[0037] Preparation Example 1

[0038] Preparation of copper curcumin derivatives

[0039] (1) Preparation of curcumin solution: Add curcumin to anhydrous ethanol, place it in a constant temperature water bath stirrer, and stir at 55℃ until curcumin is completely dissolved to obtain a transparent curcumin ethanol solution.

[0040] (2) Preparation of copper salt solution: Weigh anhydrous copper chloride in a molar ratio of copper chloride to curcumin = 1:2, dissolve it in deionized water (copper chloride: water = 1 g: 5 mL), stir until clear, and obtain an aqueous solution of copper chloride;

[0041] (3) Coordination reaction: copper chloride aqueous solution was slowly added dropwise to curcumin ethanol solution at a rate of 1-2 drops / second. After the addition was completed, the mixture was stirred at a constant temperature of 60℃ for 3 hours. During the reaction, an orange-red precipitate gradually appeared in the system.

[0042] (4) Product purification: After the reaction is completed, the reaction solution is cooled to room temperature, filtered by Buchner funnel, and the orange-red precipitate is collected.

[0043] (5) Washing and drying: Wash the precipitate 3-4 times with anhydrous ethanol (to remove unreacted raw materials and impurities), then rinse it once with deionized water. Place the washed precipitate in a vacuum drying oven and dry it at 40°C for 12 hours to obtain dried copper curcumin derivative powder.

[0044] Preparation Example 2

[0045] Preparation of peptides from Hedyotis diffusa

[0046] (1) Raw material pretreatment: The dried white flower snake tongue grass was pulverized by a pulverizer and passed through a 60-mesh sieve to obtain white flower snake tongue grass powder; the powder was added to deionized water at a material-liquid ratio of 1:20 (g:mL), and placed in an ultrasonic cleaner for ultrasonic extraction at 40℃ and 200W for 30 minutes to break the cell walls of the raw material and release the internal active substances.

[0047] (2) Enzymatic hydrolysis: Adjust the pH of the above suspension to 8.0 with 0.1 mol / L phosphate buffer, add protease at 2.0% of the dry weight of the raw material, stir to make the enzyme preparation evenly dispersed; place in a 50℃ constant temperature water bath stirrer and stir for 6 hours for enzymatic hydrolysis.

[0048] (3) Enzyme inactivation: After the enzymatic hydrolysis is completed, the system is heated to 95°C and kept at that temperature for 15 minutes to inactivate the enzyme and prevent excessive enzymatic hydrolysis from producing small molecule amino acids and losing peptide activity.

[0049] (4) Separation and purification:

[0050] 1) After enzyme inactivation, cool the system to room temperature and centrifuge at 8000 r / min for 20 min using a high-speed centrifuge. Take the supernatant and discard the lower residue. Take the centrifuged supernatant and pass it through an ultrafiltration membrane device, first through a 10 kDa cutoff membrane and then through a 1 kDa cutoff membrane for fractional filtration. Collect the 1-10 kDa cutoff liquid to obtain the crude peptide solution of Hedyotis diffusa.

[0051] 2) Add anhydrous ethanol to the crude polypeptide solution to make a final concentration of 70% v / v. After stirring evenly, let it stand at 4℃ for 12h to precipitate the residual proteins. Centrifuge again at 6000r / min for 15min and take the supernatant to obtain the white flower snake tongue polypeptide solution.

[0052] (5) Drying: The refined polypeptide liquid was placed in a vacuum freeze dryer and the parameters were set as follows: vacuum degree 10 Pa, temperature -40℃, and dried for 24 h until the system was completely dried into loose powder to obtain white flower snake tongue polypeptide powder. The polypeptide purity was 88% by HPLC.

[0053] Preparation Example 3

[0054] Preparation of polypeptide complexes

[0055] (1) Preparation of an aqueous solution of Hedyotis diffusa polypeptide:

[0056] Weigh a certain amount of Hedyotis diffusa polypeptide powder at a mass ratio of 1:1 to copper curcumin derivative and add it to a clean three-necked reaction flask; add deionized water at a concentration of 10 mg / mL, place the three-necked flask in a constant temperature water bath at 25℃, turn on the stirrer, turn on the stirrer at 400 r / min, stir for 60 min until the polypeptide powder is completely dissolved, and obtain a clear aqueous solution of Hedyotis diffusa polypeptide without flocculent precipitate.

[0057] (2) Preparation of copper curcumin derivative organic solvent solution:

[0058] Weigh out copper curcumin derivative powder at a mass ratio of 1:1 (Hedyotis diffusa polypeptide to copper curcumin derivative) and add it to a dropping funnel; add anhydrous ethanol at a concentration of 10 mg / mL; stir with a glass rod until the copper curcumin derivative is completely dissolved to obtain an orange-red transparent organic solvent solution of copper curcumin derivative.

[0059] (3) Dropwise addition of composite material and constant temperature stirring reaction:

[0060] 1) Keep the polypeptide aqueous solution stirred at 400 r / min, fix the dropping funnel above the three-necked reaction flask, and adjust the stopcock to make the copper curcumin derivative solution drop into the polypeptide aqueous solution at a rate of 1~2 drops / second.

[0061] 2) During the dropwise addition process, the system gradually turns into a uniform orange-red suspension. After the dropwise addition is completed, adjust the temperature of the constant temperature water bath to 30℃; maintain a stirring speed of 400r / min and stir at a constant temperature in the dark for 2 hours to complete the composite reaction.

[0062] 3) Transfer all the orange-red suspension after the reaction to a dialysis bag with a molecular weight cutoff of 10 kDa and tie the bag tightly; place the dialysis bag in a large amount of deionized water (dialysis fluid to suspension volume ratio ≥ 50:1) and dialyze at 25°C in the dark, changing the deionized water every 4 hours, for a total of 24 hours; transfer the suspension in the dialysis bag to a centrifuge tube and centrifuge at 10000 r / min for 15 min, discard the supernatant, and collect the lower orange-red precipitate to obtain the polypeptide complex.

[0063] Preparation Example 4

[0064] Preparation of polypeptide complexes

[0065] (1) Preparation of an aqueous solution of Hedyotis diffusa polypeptide:

[0066] Weigh a certain amount of Hedyotis diffusa polypeptide powder according to the mass ratio of Hedyotis diffusa polypeptide to copper curcumin derivative of 1:5, and add it to a clean three-necked reaction flask; add deionized water at a concentration of 10 mg / mL, place the three-necked flask in a constant temperature water bath at 25℃, turn on the stirrer, turn on the speed of 400 r / min, stir for 60 min, until the polypeptide powder is completely dissolved, and obtain a clear aqueous solution of Hedyotis diffusa polypeptide without flocculent precipitate;

[0067] (2) Preparation of copper curcumin derivative organic solvent solution:

[0068] Weigh out copper curcumin derivative powder at a mass ratio of 1:5 (Hedyotis diffusa polypeptide to copper curcumin derivative) and add it to a dropping funnel; add anhydrous ethanol at a concentration of 10 mg / mL; stir with a glass rod until the copper curcumin derivative is completely dissolved to obtain an orange-red transparent organic solvent solution of copper curcumin derivative.

[0069] (3) Drop addition and constant temperature stirring reaction: Same as preparation example 3.

[0070] Preparation Example 5

[0071] Preparation of minoxidil microspheres

[0072] (1) Preparation of oil phase: Take a dry and clean centrifuge tube, add 500 mg of lactic acid-glycolic acid copolymer; add 10 mL of mixed organic solvent (8 mL of dichloromethane and 2 mL of anhydrous ethanol), stir magnetically at room temperature for 30 min until the lactic acid-glycolic acid copolymer is completely dissolved and becomes a transparent oily liquid, add 100 mg of minoxidil to the solution, continue stirring for 15 min until the minoxidil is completely dissolved; add 10 mg of cerium dioxide, sonicate in an ice bath for 10 min at a power of 200 W, and finally obtain a uniform, transparent, particle-free oil phase liquid.

[0073] (2) Prepare the aqueous phase and emulsify to form an emulsion: Prepare a 2% polyvinyl alcohol aqueous solution: Weigh 2g of polyvinyl alcohol (PVA1788), add 100mL of deionized water, and stir at 40℃ to dissolve completely; Place 100mL of the aqueous phase in a three-necked flask, turn on the stirrer, and turn the speed to 800rpm. Slowly inject all of the oil phase from step (1) into the aqueous phase, keep stirring, and continue to emulsify at high speed for 15min to form a uniform milky white emulsion. At this time, a three-layer structure has been formed: inner layer: minoxidil, middle layer: cerium dioxide nanozyme, outer layer: lactic acid-hydroxyacetic acid copolymer coating layer.

[0074] (3) Microsphere curing: The emulsion was stirred at low speed (400 rpm) for 6 hours under room temperature, ventilation, and open conditions. After evaporation, the system was heated to 40°C and stirred for another hour to ensure complete removal of organic solvents and automatic formation of stable three-layer microspheres. It should be noted that the formation of the above three-layer structure is not a simple random distribution, but a spontaneous assembly process driven by both thermodynamics and kinetics: First, during the emulsification stage, the hydrophilic cerium dioxide nanozymes migrate to the oil-water interface under the drive of interfacial energy and accumulate on the inner side of the outer polymer layer; subsequently, during solvent evaporation and polymer curing, the hydrophobic minoxidil is encapsulated in the core region, while the nanozymes, due to their particle size and physical affinity with the polymer, are firmly embedded between the cured outer and inner layers, ultimately forming a stable three-layer structure.

[0075] (4) Finished microspheres: The solidified microsphere suspension was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the microsphere precipitate was collected; First wash: deionized water, centrifuged at 8000 rpm for 5 min; Second wash: 5% ethanol aqueous solution, centrifuged at 8000 rpm for 5 min; Third wash: deionized water, centrifuged at 8000 rpm for 5 min; Washed a total of 3 times, the microsphere precipitate was spread out and vacuum dried at 30℃ for 24 h to obtain light yellow, free-flowing minoxidil microsphere powder with a complete three-layer structure. The particle size range was determined to be 95-100 nm by dynamic light scattering method.

[0076] Preparation Example 6

[0077] Preparation of minoxidil microspheres

[0078] (1) Preparation of oil phase: Take a dry and clean centrifuge tube, add 500 mg of chitosan; add 10 mL of mixed organic solvent (8 mL of dichloromethane and 2 mL of anhydrous ethanol), stir magnetically at room temperature for 30 min until the chitosan is completely dissolved and becomes a transparent oily liquid, add 100 mg of minoxidil to the solution, continue stirring for 15 min until the minoxidil is completely dissolved; add 10 mg of cerium dioxide, sonicate in an ice bath for 10 min at a power of 200 W, and finally obtain a uniform, transparent, particle-free oil phase liquid.

[0079] (2) Prepare the aqueous phase and emulsify to form an emulsion: Prepare a 2% polyvinyl alcohol aqueous solution: Weigh 2g of polyvinyl alcohol (PVA1788), add 100mL of deionized water, stir at 40℃ to dissolve completely; Place 100mL of the aqueous phase in a three-necked flask, turn on the stirrer at 800rpm, slowly inject all of the oil phase from step (1) into the aqueous phase, keep stirring, continue high-speed emulsification for 15min to form a uniform milky white emulsion, at which point a three-layer structure prototype has been formed: inner layer: minoxidil, middle layer: cerium dioxide nanozyme, outer layer: chitosan coating layer.

[0080] (3) Microsphere curing: The emulsion is stirred at low speed under room temperature, ventilation and open conditions, and evaporated at 400 rpm for 6 hours. After evaporation, the system is heated to 40°C and stirred for another 1 hour to ensure that the organic solvent is completely removed and a stable three-layer structure microsphere is automatically formed.

[0081] (4) Finished microspheres: The solidified microsphere suspension was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the microsphere precipitate was collected; First wash: deionized water, centrifuged at 8000 rpm for 5 min; Second wash: 5% ethanol aqueous solution, centrifuged at 8000 rpm for 5 min; Third wash: deionized water, centrifuged at 8000 rpm for 5 min; Washed a total of 3 times, the microsphere precipitate was spread out and vacuum dried at 30℃ for 24 h to obtain light yellow, free-flowing minoxidil microsphere powder with a complete three-layer structure. The particle size range was determined to be 100-105 nm by dynamic light scattering method.

[0082] Example 1

[0083] Hair growth lotion

[0084] (1) Formulation composition: minoxidil microspheres (preparation example 5) 1%, film-forming agent (hydroxypropyl methylcellulose) 5%, polypeptide complex (preparation example 3) 0.5%, liquid excipient (glycerol) 5%, deionized water to make up to 100%.

[0085] (2) Preparation steps

[0086] 1) Weigh out hydroxypropyl methylcellulose according to the ratio, add a certain amount of deionized water, let it swell fully at room temperature for 2 hours, and then stir magnetically until completely homogeneous to obtain the film-forming solution;

[0087] 2) Add the specified amount of glycerol to the film-forming solution, stir magnetically at 300 r / min for 15 min, mix evenly, and obtain the intermediate product;

[0088] 3) Add minoxidil microspheres and polypeptide complex in the specified proportions to the intermediate product in sequence. Stir and disperse at 400 r / min for 20 min, add the remaining deionized water, and continue stirring at 300 r / min for 30 min until the system is homogeneous and stable to obtain hair growth liniment.

[0089] Example 2

[0090] Hair growth lotion

[0091] (1) Formulation composition: 10% minoxidil microspheres (preparation example 6), 20% film-forming agent (carbomer), 5% polypeptide complex (preparation example 4), 30% liquid excipient (butanediol), and deionized water to make up to 100%.

[0092] (2) Preparation steps

[0093] 1) Weigh out carbomer according to the ratio, add it to a measured amount of deionized water, allow it to fully swell at room temperature for 2 hours, and then stir magnetically until completely homogeneous to obtain the film-forming solution;

[0094] 2) Add the specified amount of butanediol to the film-forming solution, stir magnetically at 300 r / min for 15 min, mix evenly, and obtain the intermediate product;

[0095] 3) Add the specified amounts of minoxidil microspheres and polypeptide complex to the intermediate product in sequence. First, stir and disperse at 500 r / min for 30 min. Then, add the remaining deionized water and continue stirring at 300 r / min for 30 min until the system is homogeneous and stable to obtain the hair growth liniment.

[0096] Example 3

[0097] Hair growth lotion

[0098] (1) Formulation composition: 5% minoxidil microspheres (Preparation Example 5), 12% film-forming agent (carbomer), 2% polypeptide complex (Preparation Example 3), 15% liquid excipient (menthol), and deionized water to make up to 100%.

[0099] (2) Preparation steps

[0100] 1) Weigh out carbomer according to the ratio, add it to a measured amount of deionized water, allow it to fully swell at room temperature for 2 hours, and then stir magnetically until completely homogeneous to obtain the film-forming solution;

[0101] 2) Add the specified amount of menthol to the film-forming solution, stir magnetically at 300 r / min for 15 min, mix evenly, and obtain the intermediate product;

[0102] 3) Add minoxidil microspheres and polypeptide complex in the specified proportions to the intermediate product in sequence. Stir and disperse at 400 r / min for 20 min, add the remaining deionized water, and continue stirring at 300 r / min for 30 min until the system is homogeneous and stable to obtain hair growth liniment.

[0103] Example 4

[0104] Hair growth lotion

[0105] (1) Formulation composition: 5% minoxidil microspheres (preparation example 6), 12% film-forming agent (hydroxypropyl methylcellulose), 2% polypeptide complex (preparation example 4), 15% liquid excipient (butanediol), and deionized water to make up to 100%.

[0106] (2) Preparation steps

[0107] 1) Weigh out hydroxypropyl methylcellulose according to the ratio, add a certain amount of deionized water, let it swell fully at room temperature for 2 hours, and then stir magnetically until completely homogeneous to obtain the film-forming solution;

[0108] 2) Add the specified amount of butanediol to the film-forming solution, stir magnetically at 300 r / min for 15 min, mix evenly, and obtain the intermediate product;

[0109] 3) Add minoxidil microspheres and polypeptide complex in the specified proportions to the intermediate product in sequence. Stir and disperse at 400 r / min for 20 min, add the remaining deionized water, and continue stirring at 300 r / min for 30 min until the system is homogeneous and stable to obtain hair growth liniment.

[0110] Comparative Example 1

[0111] Hair regrowth lotion (single-layer minoxidil microspheres, no nanoenzyme layer)

[0112] (1) Formula composition:

[0113] 5% monolayer minoxidil microspheres (inner layer minoxidil, outer layer chitosan), 12% film-forming agent (carbomer), 2% polypeptide complex (same as in Example 3), 15% liquid excipient (menthol), and deionized water to make up to 100%.

[0114] (2) Preparation of minoxidil microspheres:

[0115] Minoxidil and PLGA were dissolved in an organic solvent, emulsified and solidified to obtain monolayer microspheres containing only minoxidil and chitosan.

[0116] Comparative Example 2

[0117] Hair growth lotion (without peptide complex)

[0118] (1) Formula composition:

[0119] Minoxidil microspheres (Preparation Example 5) 5%, film-forming agent (hydroxypropyl methylcellulose) 12%, liquid excipient (butanediol) 15%, deionized water to make up to 100%.

[0120] (2) Preparation method:

[0121] Follow the steps in Example 4, but without adding the polypeptide complex.

[0122] Comparative Example 3

[0123] This comparative example provides a commercially available minoxidil topical solution.

[0124] Experimental Example 1

[0125] In vitro release test

[0126] (1) Experimental samples: hair growth liniments prepared in Examples 1 to 4, comparative example 1 (single-layer microsphere liniment), and comparative example 3 (commercially available minoxidil liniment).

[0127] (2) Experimental Methods: A modified Franz diffusion cell method was used, with isolated mouse skin as the transdermal barrier. The receiving cell was filled with pH 7.4 phosphate buffer (containing 30% ethanol to increase solubility), and the temperature was maintained at (32±1)℃ with a stirring speed of 300 r / min. 2.0 g of each sample was evenly spread onto the skin surface of the supply cell. 1.0 mL samples were taken at time points (0.5, 1, 2, 4, 6, 8, 12, and 24 h), and an equal volume of isothermal receiving solution was added simultaneously. The minoxidil content was determined by HPLC, and the cumulative release percentage was calculated.

[0128] (3) Experimental results: The cumulative release rates of Examples 1 to 4 over 24 hours were 67.5%, 72.6%, 70.9%, and 69.9%, respectively, with a smooth release curve and no obvious burst release phenomenon; the cumulative release rate of Comparative Example 1 reached 89.2%, and the release in the first 2 hours exceeded 40%; the cumulative release rate of Comparative Example 3 was 95.6%, and the release was basically complete after 4 hours, indicating that the three-layer structure microspheres of this application can achieve continuous and slow release of minoxidil and effectively prolong the action time.

[0129] Experimental Example 2

[0130] Transdermal absorption test

[0131] (1) Experimental sample: Same as Experimental Example 1.

[0132] (2) Experimental methods: Ex vivo pig skin was used, and the skin was fixed in a Franz diffusion cell with the stratum corneum facing upwards. 2.0 g of each sample was evenly spread, and the skin was removed after 24 h. The stratum corneum was peeled off with adhesive tape to separate the epidermis and dermis. Minoxidil was extracted from the skin homogenate, and the amount retained in the skin was determined by HPLC; at the same time, the concentration of minoxidil in the receiving solution was determined as the transdermal amount.

[0133] (3) Experimental results: The skin retention amounts (mainly the dermis rich in hair follicles) in Examples 1-4 were 12.3, 13.1, 12.8, and 13.5 μg / cm², respectively, significantly higher than those in Comparative Example 1 (8.6 μg / cm²) and Comparative Example 3 (7.7 μg / cm²); the amount of minoxidil in the transdermal receiving solution was as follows: Figure 1 As shown, through Figure 1 The results showed that the amount of minoxidil in the transdermal receiving solution was lower in the example group than in the comparative group, indicating that the topical application of this application can effectively target and deliver minoxidil to the hair follicle site, reducing systemic absorption.

[0134] Experimental Example 3

[0135] Hair growth test

[0136] (1) Experimental samples: Examples 1-4, Comparative Examples 1-3, and control group (applied with physiological saline).

[0137] (2) Experimental Methods: Eight-week-old male C57BL / 6 mice, provided by Shanghai Medicilon Biotechnology Co., Ltd., were selected. Hair was removed from the back (2cm × 3cm). Dihydrotestosterone was injected subcutaneously into the shaved area daily for 3 consecutive weeks to establish the model. Mice were randomly divided into groups of 10. After 3 weeks of modeling, the skin on the back of the mice remained pink to the naked eye. 0.2 mL of the corresponding sample was applied to each mouse daily for 28 consecutive days. The skin in the application area was observed daily for any abnormal reactions such as erythema, edema, erosion, or roughness, and hair growth was also observed.

[0138] (3) Experimental results:

[0139] 1) Skin color change: The skin on the back of mice in the Example group began to turn black 7-10 days after administration, and was almost completely black by day 14; the blackening time for Comparative Example 1 group was 9-12 days; the blackening time for Comparative Example 2 group was 10-13 days; the blackening time for Comparative Example 3 group was 12-15 days; the control group was still pink after 28 days. It should be noted that the change in back skin color from pink to black indicates that the hair follicles have entered the growth phase.

[0140] 2) Hair growth status: On day 28, the hair growth status of Comparative Example 3 and Example 1 was as follows: Figure 2 As shown, A represents comparative group 3, and B represents example group 1. In examples 1-4, the hair in the bald area on the back of the mice had grown back well, and there was no visible skin. In comparative groups 1-3, the hair on the back of the mice was sparse and of varying lengths. In the control group, there was no obvious hair regrowth.

[0141] 3) This indicates that the hair growth lotion of this application exhibits a significant hair growth promoting effect in the androgenic alopecia model, and its effect is better than that of monolayer microspheres (Comparative Example 1) and peptide-free complex (Comparative Example 2), indicating that the three-layer microsphere structure and peptide complex have a synergistic effect in antagonizing androgen-induced hair follicle damage.

[0142] Test Example 4

[0143] Clinical trials of female androgenetic alopecia

[0144] (1) Test subjects:

[0145] 1) This study selected 120 female patients diagnosed with androgenetic alopecia who were treated at the Dermatology Clinic of the Fifth Affiliated Hospital of Anhui University of Traditional Chinese Medicine. The study was approved by the hospital's Medical Ethics Committee.

[0146] 2) Inclusion criteria: ① ≥18 years old; ② diagnosed with androgenetic alopecia by trichoscopy; ③ clinical manifestations of hair loss conforming to the Sinclair Visual Alopecia Scale grade 1-5, and the severity of hair loss conforming to the female pattern hair loss severity index 5-20; ④ patients voluntarily signed informed consent forms.

[0147] 3) Exclusion criteria: ① Patients who have used topical or systemic medications with hair growth-promoting properties in the past 12 months; ② Patients with systemic or scalp diseases that may affect hair growth or a history of acute alopecia within the past 6 months; ③ Patients with a baseline blood pressure ≤90 / 60 mmHg (1 mmHg = 0.133 kPa), or a history of orthostatic hypotension or syncope; ④ Patients with hyperkalemia of any cause, such as chronic kidney disease, hypoaldosteronism, etc.; ⑤ Patients with a history of anemia, thyroid disease, autoimmune diseases, or other systemic diseases; ⑥ Patients with a history of allergic reactions to any component of the drugs used in this study; ⑦ Patients with poor compliance who are difficult to cooperate with treatment and follow-up; ⑧ Pregnant or lactating women; ⑨ Patients who have participated in other clinical studies within the past 3 months; ⑩ Patients deemed unsuitable for participation in the study by other researchers. Patients were randomly assigned to two groups using a random ball drawing method. The control group consisted of 60 patients, aged 18–45 years (mean 34.52 ± 5.36 years) with a disease duration of 3 months to 8 years (mean 5.16 ± 1.95 years). The study group consisted of 60 patients, aged 19–46 years (mean 36.45 ± 5.28 years) with a disease duration of 4 months to 9 years (mean 4.62 ± 1.69 years). There were no statistically significant differences in general characteristics between the two groups (P > 0.05), indicating comparability.

[0148] (2) Experimental methods:

[0149] 1) The control group received a commercially available 5% minoxidil solution applied to the affected area of ​​the scalp, starting from the center of the affected area and massaging for 3-5 minutes, 1 ml (containing 50 mg of minoxidil) per application, once a day. The study group received the same treatment as in Example 4, applying the same amount once a day. Both groups continued treatment for 12 months.

[0150] 2) Observation indicators and evaluation criteria:

[0151] a. Clinical efficacy: Trichoscopy was used to evaluate the change in average hair density per unit area in the occipital and central frontal regions from baseline at 3, 6, 9, and 12 months of drug treatment. The average hair density at 12 months was the primary endpoint.

[0152] b. Safety: Record adverse events, including scalp itching, allergic reactions, dizziness, breast tenderness, menstrual disorders, hyperkalemia, abnormal blood pressure, gastrointestinal reactions (nausea, vomiting, etc.), and transient increases in plasma creatinine and blood urea nitrogen, and calculate the incidence rate.

[0153] 3) Statistical methods: SPSS 24.0 statistical software was used for data analysis. Normally distributed continuous data were expressed as mean ± standard deviation. Data expressed as mean ± standard deviation (s) were analyzed using a t-test; count data expressed as [n (%)] were analyzed using a χ² test. 2 The test was performed, and P < 0.05 was considered statistically significant.

[0154] (3) Experimental data: The average hair density of the two groups is shown in Table 1, and the safety comparison is shown in Table 2;

[0155] Table 1 Comparison of clinical efficacy between the two groups (fu / cm) 2 , ±s)

[0156]

[0157] Table 2. Comparison of safety between the two groups [n (%)]

[0158]

[0159] (4) Results: Table 1 shows that the average hair density in the study group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Table 2 shows that the total incidence of adverse events in the study group was only 1 case higher than that in the control group, and its safety was almost the same as that of the commercially available control group.

[0160] In summary, this application solves the technical challenges of poor transdermal absorption, low bioavailability, and single-action effects of traditional minoxidil formulations through the synergistic design of a three-layered minoxidil microsphere structure and a peptide complex. Experimental results show that the three-layered microsphere structure achieves slow, controlled release of minoxidil; transdermal absorption assays confirm that this formulation can target the drug to the hair follicle site, with a skin retention of 12.3–13.5 μg / cm², which is 1.5–1.7 times that of commercially available formulations, reducing systemic absorption; and in an animal model of androgenetic alopecia, the hair growth-promoting effect of this application is significantly superior to that of monolayered microsphere formulations, formulations without peptide complexes, and commercially available products, verifying the synergistic effect of the three-layered microsphere structure and the peptide complex. It has good clinical application prospects, and clinical trials showed that its hair density is higher than that of commercially available products, while its safety is almost identical.

[0161] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A hair growth lotion, characterized in that, The hair growth liniment comprises, by weight percentage: 1-10% minoxidil microspheres, 5-20% film-forming agent, 0.5-5% polypeptide complex, 5-30% liquid excipients, and the remainder being deionized water.

2. The hair growth lotion as described in claim 1, characterized in that, The minoxidil microspheres have a three-layer structure: the inner layer is minoxidil, the middle layer is nanozyme, and the outer layer is any one of lactic acid-glycolic acid copolymer or chitosan.

3. The hair growth lotion as described in claim 2, characterized in that, The nanozyme is cerium dioxide.

4. The hair growth lotion as described in claim 1, characterized in that, The polypeptide complex is a complex of Hedyotis diffusa polypeptide and copper curcumin derivative.

5. The hair growth lotion as described in claim 4, characterized in that, The mass ratio of the Hedyotis diffusa polypeptide to the copper curcumin derivative is 1:1~5.

6. The hair growth lotion as described in claim 1, characterized in that, The film-forming agent is either hydroxypropyl methylcellulose or carbomer.

7. The hair growth lotion as described in claim 1, characterized in that, The liquid excipient is any one of glycerin, butylene glycol, or menthol.

8. A method for preparing a hair growth liniment, characterized in that, The preparation method includes the following steps: (1) Add the film-forming agent to deionized water, allow it to swell fully and stir until homogeneous to obtain the film-forming solution; (2) Add the liquid excipients to the film-forming solution and stir to mix evenly to obtain the intermediate product; (3) Add minoxidil microspheres and polypeptide complex to the intermediate in sequence, stir and disperse evenly, add deionized water, and continue stirring until the system is homogeneous and stable to obtain hair growth liniment.

9. The method for preparing a hair growth liniment as described in claim 8, characterized in that, The method for preparing the minoxidil microspheres includes the following steps: (1) Minoxidil and polymer materials are dissolved in an organic solvent, cerium dioxide nanozyme is added and dispersed evenly to obtain an oil phase; (2) The oil phase is added to the aqueous phase containing the emulsifier and emulsified to form an emulsion; (3) Stirring to evaporate the organic solvent and solidify the microspheres; (4) Centrifuge, wash and dry to obtain a three-layer structure of minoxidil microspheres with an inner layer of minoxidil, a middle layer of cerium dioxide nanozyme and an outer layer of lactic acid-hydroxyacetic acid copolymer or chitosan.

10. The method for preparing a hair growth liniment as described in claim 8, characterized in that, The preparation method of the polypeptide complex includes the following steps: (1) Dissolve the white flower snake tongue grass polypeptide in deionized water to obtain a polypeptide solution; (2) Dissolve the copper curcumin derivative in an organic solvent to obtain a copper curcumin derivative solution; (3) Add the copper curcumin derivative solution dropwise to the polypeptide solution, stir, purify and dry to obtain the polypeptide complex.

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