A lyophilized powder composition for hair regrowth and a method of preparing the same

Abstract

The present application relates to the technical field of freeze-dried powder, and in particular to a freeze-dried powder composition for hair regeneration and a preparation method thereof. The freeze-dried powder composition comprises 5-15 parts of vascular endothelial growth factor, 3-10 parts of platelet-derived growth factor, 1-5 parts of copper peptide, 0.5-3 parts of acetyl tetrapeptide-3, 2-8 parts of baicalein, 1-5 parts of asiaticoside, 20-40 parts of freeze-drying protectant, and 1-3 parts of nano-silicon dioxide. The preparation method comprises mixing the pretreated vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside with the freeze-drying protectant and nano-silicon dioxide, and then freeze-drying. The freeze-dried powder composition can promote the growth of hair, improve the microenvironment of the scalp, and provide long-lasting support for the healthy growth of hair. The preparation method improves the stability and biological activity of the active ingredients, and enhances the overall performance and safety of the product.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of freeze-dried powder, in particular to a freeze-dried powder composition for hair regeneration and a preparation method thereof. BACKGROUND

[0002] In recent years, due to environmental, work pressure, genetic and other factors, the number of people with hair loss is increasing. Hair loss is caused by many internal factors, such as aging or hormonal imbalance, as well as various diseases and severe burns or wounds, etc., which is usually accompanied by thinning of the skin in the bald area, and these symptoms are caused by loss of follicle function.

[0003] Currently, there are several ways to treat hair loss, such as oral or topical medications, or hair transplantation. However, these treatment methods have limitations, and drug treatment can only temporarily alleviate symptoms. For example, oral finasteride has sexual side effects and can cause malformations, and after stopping medication, hair loss will accelerate. Hair transplantation is a reliable surgical treatment method, but the survival rate of transplants is low and the cost is high, and there is a certain risk.

[0004] With the development of society, people pay more and more attention to hair that has protective and aesthetic effects. Research on the periodic regeneration of hair follicles has practical and important significance.

[0005] Hair regeneration is one of the important research directions in the field of medical cosmetology and skin health management in recent years. With the increasing demand for health and beauty, the development of efficient and safe hair regeneration products has become a research hotspot. However, the process of hair regeneration is complex and involves the synergistic action of multiple bioactive factors, which play an important role in promoting hair follicle growth, repairing damaged hair follicles, and improving scalp microcirculation.

[0006] Currently, freeze-drying technology is widely used in the preparation of hair regeneration products due to its ability to effectively preserve the activity of bioactive substances. Freeze-drying technology avoids the destruction of active ingredients by high temperature through low-temperature drying, while improving the stability and storage life of the product. However, traditional freeze-drying protectants such as trehalose and mannitol can protect bioactive substances to some extent, but their protective effect under certain conditions is limited, and they may affect the bioavailability of the final product.

[0007] Despite the progress made in existing technology, there are still some problems that need to be solved. For example, how to further improve the stability and bioavailability of bioactive substances; how to optimize the formula of freeze-drying protectants to adapt to different types of bioactive substances. The solution to these problems will help to develop more efficient and safer hair regeneration products, meet market demand and promote the development of related technologies. SUMMARY

[0008] To solve the above problems, the application provides a lyophilized powder composition for hair regeneration and a preparation method thereof. The lyophilized powder composition has good stability, realizes efficient hair regeneration, and significantly improves scalp health. It not only promotes hair growth, but also fundamentally improves the scalp microenvironment, providing lasting support for the healthy growth of hair. The preparation method improves the stability and biological activity of active ingredients through a pretreatment process, optimizes the uniformity and solubility of the composition, and thus improves the overall performance and safety of the product. In the lyophilization process, multi-stage temperature rise is adopted, which can make the water sublimate uniformly and avoid the problem of local crystallization unevenness.

[0009] The technical scheme adopted by the application to achieve the above-mentioned purposes is as follows:

[0010] A lyophilized powder composition for hair regeneration, comprising the following components by weight fraction:

[0011] 5-15 parts of vascular endothelial growth factor, 3-10 parts of platelet-derived growth factor, 1-5 parts of copper peptide, 0.5-3 parts of acetyl tetrapeptide-30, 2-8 parts of baicalein, 1-5 parts of asiaticoside, 20-40 parts of lyophilization protectant, 1-3 parts of nano silicon dioxide;

[0012] The lyophilization protectant comprises the following components by weight fraction: modified trehalose 25-35 parts, modified mannitol 10-20 parts, hydroxypropyl-beta-cyclodextrin 15-25 parts, pegylated phospholipid 5-8 parts, and L-proline 8-10 parts;

[0013] The preparation method of the modified trehalose comprises the following steps:

[0014] Step a: trehalose and sodium tripolyphosphate are reacted at pH 9.4-9.6 and 50-55°C for 4-5 hours, then ion exchange purification after dialysis to obtain product 1;

[0015] Step b: product 1 and palmitoyl chloride are reacted in argon-protected tetrahydrofuran at 26-30°C for 2-3 hours, and then supercritical CO2 extraction purification is performed to obtain product 2;

[0016] Step c: 100 parts by weight of product 2 and 220-260 parts by weight of mPEG-NHS are reacted in pH 7.2-7.6 PBS at 2-8°C for 10-14 hours, then tangential flow filtration and lyophilization are performed to obtain the product.

[0017] Preferably, in step a, the weight ratio of trehalose to sodium tripolyphosphate is 100:58-75.

[0018] Preferably, in step b, the weight ratio of product 1 to palmitoyl chloride and tetrahydrofuran is 100:45-50:300-500.

[0019] Preferably, the weight ratio of product 2 to mPEG-NHS in step c is 100: 100-150.

[0020] Trehalose reacts with sodium tripolyphosphate under alkaline conditions to form phosphorylated trehalose. The introduction of phosphate groups increases the negative charge density of trehalose, making it more water-soluble and ion-exchangeable. In step b, esterification occurs between the unreacted hydroxyl group (-OH) of trehalose and palmitoyl chloride (C 15 H 31 COCl) to form a palmitate bond. The introduction of the palmitate bond changes the molecular structure of trehalose, transforming it from a hydrophilic molecule to a hydrophobic amphiphilic molecule. The hydrophobic end of the amphiphilic modified trehalose (palmitoyl chain) adsorbs on the ice crystal surface, and the hydrophilic end (phosphorylated / PEGylated part) binds with water molecules to form a "molecular shield": modified trehalose limits ice crystal size through steric hindrance, reducing mechanical damage to proteins and improving the activity retention rate of growth factors. The hydrophobic chain binds with the hydrophobic region of the protein, preventing denaturation of the protein-water interface during lyophilization. The hydrophobic chain can also wrap and protect other active ingredients, preventing their aggregation or inactivation during the lyophilization process. The palmitoyl chain forms a network structure with L-proline and hydroxypropyl-β-cyclodextrin through hydrophobic-hydrophobic interaction, increasing the glass transition temperature, avoiding the collapse of amorphous phase during lyophilization, enhancing mechanical strength, and preventing lyophilized powder from cracking. At the same time, the palmitoyl chain inserts into the hydrophobic cavity of cyclodextrin to form a "supramolecular carrier", improving the solubility of baicalein. The amphiphilic modified trehalose is directionally arranged at the solid-liquid interface during reconstitution, reducing surface tension and shortening reconstitution time. By reacting with mPEG-NHS (methoxy polyethylene glycol-succinimidyl ester), polyethylene glycol chains (PEG chains) are further introduced onto the trehalose molecule. PEG chains have good biocompatibility and water solubility, which can further improve the stability and dispersibility of trehalose, effectively prevent the aggregation of growth factors, and help uniformly wrap and protect other active ingredients.

[0021] Preferably, the preparation method of the modified mannitol is as follows: comprising the following steps:

[0022] Step A: Mix mannitol and succinic anhydride, add 4-dimethylaminopyridine and solvent, and react at 50-55°C under nitrogen atmosphere for 5-6 hours;

[0023] Step B: The reaction solution is distilled under reduced pressure to remove the solvent, precipitated with diethyl ether, and then purified by ultrafiltration;

[0024] Step C: Spray drying at an inlet temperature of 70-75°C and an outlet temperature of 50-55°C to obtain the product.

[0025] Preferably, in step A, the molar ratio of mannitol to succinic anhydride to 4-dimethylaminopyridine is 1:0.25-0.35:0.03-0.08, the solvent is DMF or DMSO, and the amount used is 50-100 mL of DMF or DMSO per mole of mannitol.

[0026] By reacting with succinic anhydride, a new functional group (such as carboxyl) is introduced into the molecular structure of mannitol, which increases the polarity of the molecule, improves the water solubility, reduces the crystallinity, forms an amorphous structure, and avoids "clumping" when the lyophilized powder is reconstituted. This modified mannitol can better dissolve and disperse in the lyophilization protectant, thereby improving the uniformity of the entire composition. At the same time, the carboxylic acid group can form hydrogen bonds with the amino group of the protein, reducing the freeze-drying damage. In cooperation with L-proline and hydroxypropyl-β-cyclodextrin, the stability of the glassy matrix is enhanced. In cooperation with modified trehalose, the carboxylic acid group forms an electrostatic complex with phosphorylated trehalose, optimizing the lyophilized powder porosity and specific surface area. In combination with nano-silicon dioxide, the carboxylic acid group anchors the silicon dioxide surface through hydrogen bonds, enhancing drug dispersibility. Carboxyl groups can also improve compatibility with the stratum corneum and promote hair follicle penetration.

[0027] The above-mentioned method for preparing a lyophilized powder composition for hair regeneration comprises pretreating vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside; mixing modified trehalose, hydroxypropyl-β-cyclodextrin, pegylated phospholipid, L-proline, and modified mannitol to obtain a lyophilization protectant; and then mixing the pretreated vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside with the lyophilization protectant and nano-silicon dioxide, and lyophilizing to obtain the lyophilized powder composition.

[0028] Preferably, the pretreatment comprises dissolving the vascular endothelial growth factor and the platelet-derived growth factor in a PBS buffer containing 0.1% human serum albumin, and storing at 2-8°C in the dark, wherein the weight / volume ratio of the total mass of the vascular endothelial growth factor and the platelet-derived growth factor to the PBS buffer is 1 mg:10-12 ml.

[0029] HSA (human serum albumin) as a carrier protein can reduce the adsorption loss of growth factors on the container wall, provide colloidal protection, and inhibit the aggregation of VEGF / PDGF. The PBS buffer (pH 7.4) provides a stable ionic environment to prevent protein denaturation. HSA cooperates with the subsequently added lyophilization protectant (such as modified trehalose) to form a "protein-sugar" complex, reducing ice crystal damage.

[0030] Preferably, the pretreatment comprises dissolving copper peptide and acetyl tetrapeptide-3 in an aqueous solution containing 1-2% propylene glycol, 0.05% ethylenediaminetetraacetic acid and 0.05% Tween 80, and the weight / volume ratio of the total weight of copper peptide and acetyl tetrapeptide-3 to the aqueous ethylenediaminetetraacetic acid solution is 1 mg: 1-2 ml.

[0031] Propylene glycol enhances the solubility of copper peptide, EDTA chelates metal ions (such as Cu 2+ ), prevents oxidative degradation of polypeptides, inhibits enzymatic hydrolysis and aggregation, and Tween 80 coats polypeptides to form micelles, avoiding hydrophobic aggregation between polypeptide molecules during the freeze-drying process. Propylene glycol and Tween 80 synergistically destroy the lipid barrier of the stratum corneum, improving the transdermal efficiency of the subsequent freeze-dried powder.

[0032] Preferably, the pretreatment comprises adding baicalein and asiaticoside to hydroxypropyl-β-cyclodextrin in an aqueous ethanol solution, stirring at 38-42℃ for 1.5-2.5 hours, wherein the amount of hydroxypropyl-β-cyclodextrin is 0.9-1.1 times the total amount of baicalein and asiaticoside, and the weight / volume ratio of hydroxypropyl-β-cyclodextrin to the aqueous ethanol solution is 0.5: 10-15.

[0033] Hydroxypropyl-β-cyclodextrin has a unique cavity structure and can form inclusion complexes with baicalein and asiaticoside, significantly improving their solubility in water. The formation of inclusion complexes can protect baicalein and asiaticoside from oxidation and degradation, improving their stability during freeze-drying and storage. The formation of inclusion complexes helps to improve the bioavailability of baicalein and asiaticoside, so that they can play a more effective role when used.

[0034] The present application has the following beneficial effects:

[0035] The freeze-dried powder composition of the present application contains a plurality of key growth factors (such as vascular endothelial growth factor, platelet-derived growth factor) and bioactive peptides (such as copper peptide, acetyl tetrapeptide-3), which can synergistically promote the proliferation and differentiation of hair follicle cells, induce the transformation of hair follicles from the resting phase to the growth phase, and significantly improve the regenerative ability of hair. Natural plant ingredients such as baicalein and asiaticoside have antioxidant and anti-inflammatory effects, which can improve the scalp microenvironment and reduce the inflammatory response around the hair follicle, creating favorable conditions for hair growth. The modified trehalose, modified mannitol, hydroxypropyl-beta-cyclodextrin, pegylated phospholipids and L-proline in the freeze-drying protectant can form a stable glassy structure to prevent the denaturation and inactivation of active ingredients during freeze-drying and storage, significantly extending the shelf life of the product. Nano-silicon dioxide can be adsorbed on the surface of the active ingredients to prevent aggregation and precipitation, further improving the stability and solubility of the freeze-dried powder. The freeze-dried powder composition has good stability, realizes efficient hair regeneration, and significantly improves scalp health. It not only promotes hair growth, but also fundamentally improves the scalp microenvironment, providing lasting support for healthy hair growth.

[0036] The freeze-dried powder composition for hair regeneration provided by the present application combines two growth factors, wherein the vascular endothelial growth factor promotes angiogenesis around the hair follicle, improves the blood supply of the hair papilla, and provides sufficient nutrition for the hair follicle; the platelet-derived growth factor directly stimulates the activation of hair follicle stem cells, activates hair follicle stem cell proliferation, and promotes hair growth; the two synergistically extend the growth phase of the hair follicle, promote the proliferation and differentiation of hair follicle cells, and induce the transformation of the hair follicle from the resting phase to the growth phase. In the polypeptide synergistic system, copper peptide promotes hair follicle growth, antioxidant and anti-inflammatory, and repairs damaged tissue, providing a good environment for healthy hair growth; and acetyl tetrapeptide-3 further improves the scalp condition through anti-inflammatory and skin tightening effects, promoting hair growth. The combination of the two can more comprehensively improve the effect of hair regeneration. Natural ingredients such as baicalein and asiaticoside have antioxidant and anti-inflammatory effects, which can improve the scalp environment and reduce the inflammatory response around the hair follicle, thereby creating favorable conditions for hair growth.

[0037] Trehalose is a natural non-reducing disaccharide with good stability. Modified trehalose, through the introduction of phosphate groups, palmitoyl groups and PEG chains, triple modification, realizes the synergistic effect of charge stability, interfacial activity and space protection, significantly improves its performance in lyophilized protectant. Modified trehalose not only improves its own chemical stability and thermal stability, but also improves its solubility and dispersibility, and enhances its protection ability to other active ingredients. These characteristics make modified trehalose play an important protective and synergistic role in hair regeneration lyophilized powder composition. The hydrogen bond between the phosphate group and the hydroxyl group of cyclodextrin enhances the inclusion stability of baicalein and other active ingredients. Together with L-proline, it forms a protective layer to reduce the dehydration damage of proteins. During the lyophilization process, modified trehalose can form a stable glassy structure to prevent the denaturation and inactivation of active ingredients. The hydroxypropyl-β-cyclodextrin and polyethylene glycol phospholipid in the lyophilized protectant can form inclusion compounds or complexes with growth factors and other active ingredients, further improving their stability. This synergistic protection makes the lyophilized powder maintain high biological activity during storage and transportation. Nano-silicon dioxide has good dispersibility and adsorbability, which can be adsorbed on the surface of active ingredients to prevent aggregation and precipitation. This not only improves the solubility of lyophilized powder, but also prolongs its shelf life.

[0038] Modified mannitol significantly improves its solubility, stability and biocompatibility by introducing new functional groups through specific chemical reactions. In the lyophilized protectant, modified mannitol can better protect other active ingredients, improve the overall performance and safety of the product.

[0039] The preparation method of the present application not only improves the stability and biological activity of the active ingredients, but also optimizes the uniformity and solubility of the composition, thereby improving the overall performance and safety of the product. In addition, the pretreatment process also simplifies the production process, improves the production efficiency and repeatability. In the lyophilization process, multi-stage stepwise heating is adopted, which can make the water sublimate uniformly and avoid the problem of local crystallization unevenness. This process can improve the uniformity and stability of the lyophilized powder. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0041] Vascular endothelial growth factor brand: source leaf, platelet-derived growth factor-recombinant human platelet-derived growth factor BB (PDGF-BB), brand, Abbkine; copper peptide, Hubei Xiyu Hong Biological Medicine Technology Co., Ltd.; acetyl tetrapeptide-3, Xi'an Shilin Biological Technology Co., Ltd.; EDTA, Beijing Aomeijia Medicine Technology Co., Ltd.; baicalin, effective ingredient content 98%, Yangling Ciyuan Biological Technology Co., Ltd.; asiaticoside, 80 mesh, Lanzhou Waterless Biological Technology Co., Ltd.; hydroxypropyl-β-cyclodextrin, Hubei Yamide Biological Medicine Co., Ltd.; nano silicon dioxide, 50-80nm; trehalose, Zhejiang Binmei Biological Technology Co., Ltd.; mannitol, Xi'an Hongyao Pharmaceutical Auxiliary Material Co., Ltd.; L-proline, Anhui Zhonghong Biological Engineering Co., Ltd.; sodium tripolyphosphate, Hunan Xunao Biological Engineering Co., Ltd.; palmitoyl chloride, Condis Chemical Industry; PBS buffer, Shanghai Zeye Biological Technology Co., Ltd.; human serum albumin, brand: Targetmol; pegylated phospholipid, DSPE-PEG2000, purity 95%, Shaanxi Xingbei Aike Biological Technology Co., Ltd.; MPEG-NHS (methoxy polyethylene glycol active ester) molecular weight 2000, Shanghai source leaf biological technology Co., Ltd.; succinic anhydride, Wuhan Smack Biological Technology Co., Ltd. The raw materials used in the following examples are all ordinary commercially available products.

[0042] Example 1

[0043] A lyophilized powder composition for hair regeneration, comprising the following components by weight fraction:

[0044] Vascular endothelial growth factor 10 parts, platelet-derived growth factor 6 parts, copper peptide 3 parts, acetyl tetrapeptide-3 2 parts, baicalin 5 parts, asiaticoside 3 parts, lyophilization protective agent 30 parts, nano silicon dioxide 2 parts;

[0045] The lyophilization protective agent comprises the following components by weight fraction: modified trehalose 28 parts, modified mannitol 15 parts, hydroxypropyl-β-cyclodextrin 20 parts, pegylated phospholipid 6 parts, L-proline 9 parts;

[0046] The preparation method of the modified trehalose comprises the following steps:

[0047] Step a, 100 parts by weight of trehalose was reacted with 65 parts by weight of sodium tripolyphosphate at pH 9.4-9.6, 53℃ for 4.5 hours, the pH was adjusted to 7.0±0.2 with 1M HCl to terminate the reaction, two-stage dialysis was carried out in turn using a dialysis bag with a molecular weight cut-off of 3.5kDa: the first stage: dialysis with 0.1M NaCl solution, volume ratio 1:20, 4℃ for 4 hours; the second stage: dialysis with ultrapure water, volume ratio 1:50, 4℃ for 24 hours; the dialysate was loaded onto a DEAE cellulose column, eluted with a linear gradient of 0-0.5M NaCl, flow rate 1mL / min; the electric elution fractions were collected, concentrated by rotary evaporation to a solid content ≥20%, and freeze-dried to obtain product 1;

[0048] Step b, 100 parts by weight of product 1 was reacted with 47 parts by weight of palmitoyl chloride in 400 parts of tetrahydrofuran under argon protection at 28℃ for 2.5 hours, the reaction solution was added dropwise into 10 times volume of -20℃ methanol for precipitation, centrifuged at 8000rpm for 15 minutes, and the precipitate was collected; supercritical CO2 extraction purification was carried out under the following conditions: pressure 80bar, temperature 35℃; CO2 flow rate 8L / min; cycle extraction 3 times, each for 30 minutes; vacuum drying at 40℃ for 24 hours, vacuum degree ≤5Pa, to obtain product 2;

[0049] Step c, 100 parts by weight of product 2 was reacted with 240 parts by weight of mPEG-NHS in PBS at pH 7.2-7.6 at 5℃ for 12 hours, purified by a 10kDa tangential flow filtration system, and freeze-dried after tangential flow filtration, with the freeze-drying process being: pre-freezing at -80℃ for 2 hours, drying at -40℃ / 15Pa for 20 hours, and secondary drying at 25℃ / 5Pa for 6 hours.

[0050] The preparation method of the modified mannitol comprises the following steps:

[0051] Step A, mannitol, succinic anhydride and 4-dimethylaminopyridine were added to DMF, and reacted under a nitrogen atmosphere at 52℃ for 5.5 hours, with the molar ratio of mannitol, succinic anhydride and 4-dimethylaminopyridine being 1:0.3:0.05, and the amount of DMF being 75mL per mole of mannitol;

[0052] Step B, the reaction solution was distilled under reduced pressure to remove the solvent, precipitated with diethyl ether, and then purified by ultrafiltration;

[0053] Step C, spray drying, inlet temperature 72℃, outlet temperature 52℃, to obtain the product.

[0054] The preparation method of the freeze-dried powder composition for hair regeneration comprises the following steps:

[0055] Step 1, pretreatment: VEGF, PDGF are dissolved in PBS buffer solution containing 0.1% HSA (pH 7.2-7.6), stored at 6℃ in the dark, the total mass of VEGF, PDGF and the weight volume ratio of PBS buffer solution is 1mg:11ml; Cu peptide and acetyl tetrapeptide-3 are dissolved in an aqueous solution containing 1.5% propylene glycol, 0.05% EDTA (ethylenediamine tetraacetic acid) and 0.05% Tween 80, the weight volume ratio of Cu peptide and acetyl tetrapeptide-3 and the EDTA aqueous solution is 1mg:1ml; Baicalein and asiaticoside are added to an ethanol aqueous solution with an ethanol concentration of 40%, stirred at 40℃ for 30 minutes, then diluted to an ethanol concentration of 20% by adding water, then hydroxypropyl-β-cyclodextrin is added and stirred at 40℃ for 2 hours, then rotary evaporation is performed to remove ethanol, wherein the amount of hydroxypropyl-β-cyclodextrin is 1:1 of the total amount of baicalein and asiaticoside, and the weight volume ratio of the total amount of baicalein and asiaticoside and the initial 40% ethanol aqueous solution is 1g:12ml;

[0056] Step 2, modified trehalose and hydroxypropyl-β-cyclodextrin are dissolved in a 5% ethanol aqueous solution at 45℃, the weight ratio of modified trehalose and the ethanol aqueous solution is 1:18, then polyglycolated phospholipid, L-proline and modified mannitol are sequentially added, and mixed at 30℃ to obtain a freeze-drying protective agent;

[0057] Step 3, nano-silicon dioxide is added to the freeze-drying protective agent, and after ultrasonic dispersion (150W, 18 minutes), the pretreated VEGF, Cu peptide and acetyl tetrapeptide-3, baicalein and asiaticoside of step 1 are added and mixed evenly; Step 4, the mixed solution obtained in step 3 is kept at 4℃ for 30 minutes, cooled to -80℃ for 2 hours, then dried at -48℃, 12Pa for 30 hours, and then dried at 22℃, 3Pa for 10 hours; ground through a 100 mesh sieve, and packaged with nitrogen.

[0058] Example 2

[0059] A freeze-dried powder composition for hair regeneration, comprising the following components by weight fraction:

[0060] VEGF 15 parts, PDGF 3 parts, Cu peptide 5 parts, acetyl tetrapeptide-3 0.5 parts, baicalein 8 parts, asiaticoside 1 part, freeze-drying protective agent 40 parts, nano-silicon dioxide 1 part;

[0061] The freeze-drying protective agent comprises the following components by weight fraction: modified trehalose 35 parts, modified mannitol 10 parts, hydroxypropyl-β-cyclodextrin 15 parts, polyglycolated phospholipid 8 parts, L-proline 8 parts;

[0062] The preparation method of the modified trehalose comprises the following steps:

[0063] Step a, 100 parts by weight of trehalose was reacted with 75 parts by weight of sodium tripolyphosphate at pH 9.4-9.6, 55℃ for 4 hours, and purified (same as Example 1) to obtain product 1;

[0064] Step b, 100 parts by weight of product 1 was reacted with 50 parts by weight of palmitoyl chloride in 300 parts of tetrahydrofuran under argon protection at 26℃ for 3 hours, and purified by supercritical CO2 extraction (same as Example 1) to obtain product 2;

[0065] Step c, 100 parts by weight of product 2 was reacted with 260 parts by weight of mPEG-NHS in PBS at pH 7.2-7.6 at 2℃ for 14 hours, and freeze-dried after tangential flow filtration (same as Example 1). The freeze-drying process was as follows: pre-freezing at -80℃ for 2 hours, drying at -40℃ / 15 Pa for 20 hours, and secondary drying at 25℃ / 5 Pa for 6 hours.

[0066] The preparation method of the modified mannitol is as follows: comprising the following steps:

[0067] Step A, mannitol, succinic anhydride and 4-dimethylaminopyridine were added to DMF, and the reaction was carried out at 55℃ for 5 hours under nitrogen atmosphere, the molar ratio of mannitol, succinic anhydride and 4-dimethylaminopyridine was 1:0.25:0.03, and the amount of DMF was 50 mL per mole of mannitol;

[0068] Step B, the reaction solution was distilled under reduced pressure to remove the solvent, precipitated with diethyl ether, and purified by ultrafiltration;

[0069] Step C, spray drying, inlet temperature 75℃, outlet temperature 55℃, to obtain

[0070] The preparation method of the above freeze-dried powder composition for hair regeneration comprises the following steps:

[0071] Step 1, pretreatment: VEGF, PDGF are dissolved in PBS buffer solution containing 0.1% HSA (pH 7.2-7.6), stored at 4℃ in the dark, the total mass of VEGF, PDGF and the weight volume ratio of PBS buffer solution is 1mg:12ml; Cu peptide and acetyl tetrapeptide-3 are dissolved in an aqueous solution containing 1% propylene glycol, 0.05% EDTA (ethylenediamine tetraacetic acid) and 0.05% Tween 80, the weight volume ratio of Cu peptide and acetyl tetrapeptide-3 and the EDTA aqueous solution is 1mg:2ml; Baicalein and asiaticoside are added to an ethanol aqueous solution with an ethanol concentration of 40%, stirred at 40℃ for 30 minutes, then diluted to an ethanol concentration of 20% by adding water, then hydroxypropyl-β-cyclodextrin is added and stirred at 38℃ for 2.5 hours, then rotary evaporation is performed to remove ethanol, wherein the amount of hydroxypropyl-β-cyclodextrin is 0.9 times the total amount of baicalein and asiaticoside, and the weight volume ratio of the total amount of baicalein and asiaticoside and the initial 40% ethanol aqueous solution is 1g:15ml;

[0072] Step 2, modified trehalose and hydroxypropyl-β-cyclodextrin are dissolved in a 5% ethanol aqueous solution at 40℃, the weight ratio of modified trehalose and the ethanol aqueous solution is 1:15, then polyglycolated phospholipid, L-proline and modified mannitol are sequentially added, and mixed at 28℃ to obtain a freeze-drying protective agent;

[0073] Step 3, nano-silicon dioxide is added to the freeze-drying protective agent, and after ultrasonic dispersion (100W, 20 minutes), VEGF, Cu peptide and acetyl tetrapeptide-3, baicalein and asiaticoside pretreated in step 1 are added and mixed evenly; Step 4, the mixed solution obtained in step 3 is kept at 4℃ for 30 minutes, cooled to -80℃ for 2 hours, then dried at -45℃, 15Pa for 36 hours, and then dried at 25℃, 5Pa for 8 hours; ground through a 120 mesh sieve, and packaged with nitrogen, to obtain the freeze-dried powder composition for hair regeneration.

[0074] Example 3

[0075] A freeze-dried powder composition for hair regeneration, comprising the following components by weight fraction:

[0076] VEGF 5 parts, PDGF 10 parts, Cu peptide 1 part, acetyl tetrapeptide-3 3 parts, baicalein 2 parts, asiaticoside 5 parts, freeze-drying protective agent 20 parts, nano-silicon dioxide 3 parts;

[0077] The freeze-drying protective agent comprises the following components by weight fraction: modified trehalose 25 parts, modified mannitol 20 parts, hydroxypropyl-β-cyclodextrin 25 parts, polyglycolated phospholipid 5 parts, L-proline 10 parts;

[0078] The preparation method of the modified trehalose comprises the following steps:

[0079] Step a, 100 parts by weight of trehalose was reacted with 58 parts by weight of sodium tripolyphosphate at pH 9.4-9.6, 50℃ for 5 hours, purified (same as Example 1), to obtain product 1;

[0080] Step b, 100 parts by weight of product 1 was reacted with 45 parts by weight of palmitoyl chloride in 500 parts of tetrahydrofuran under argon protection at 30℃ for 2 hours, purified by supercritical CO2 extraction (same as Example 1), to obtain product 2;

[0081] Step c, 100 parts by weight of product 2 was reacted with 220 parts by weight of mPEG-NHS in PBS at pH 7.2-7.6 at 8℃ for 10 hours, freeze-dried after tangential flow filtration (same as Example 1), the freeze-drying process was: pre-freezing at -80℃ for 2 hours, drying at -40℃ / 15Pa for 20 hours, secondary drying at 25℃ / 5Pa for 6 hours, to obtain the product.

[0082] The preparation method of the modified mannitol is as follows: comprising the following steps:

[0083] Step A, mannitol, succinic anhydride and 4-dimethylaminopyridine were added into DMF, and reacted at 50℃ for 6 hours under nitrogen atmosphere, the molar ratio of mannitol, succinic anhydride and 4-dimethylaminopyridine was 1:0.35:0.08, and the amount of DMF was 100 mL per mole of mannitol;

[0084] Step B, the reaction solution was distilled under reduced pressure to remove the solvent, precipitated with diethyl ether and purified by ultrafiltration;

[0085] Step C, spray drying, inlet temperature 70℃, outlet temperature 50℃, to obtain the product.

[0086] The preparation method of the above freeze-dried powder composition for hair regeneration, comprising the following steps:

[0087] Step 1, pretreatment: VEGF, PDGF are dissolved in PBS buffer solution (pH 7.2-7.6) containing 0.1% human serum albumin (HSA) and stored at 8℃ in the dark, the weight / volume ratio of VEGF, PDGF and PBS buffer solution is 1 mg:10 ml; copper peptide and acetyl tetrapeptide-3 are dissolved in an aqueous solution containing 2% propylene glycol, 0.05% EDTA (ethylenediaminetetraacetic acid) and 0.05% Tween 80, the weight / volume ratio of copper peptide and acetyl tetrapeptide-3 and the EDTA aqueous solution is 1 mg:1.5 ml; baicalein and asiaticoside are added to an aqueous solution of ethanol with an ethanol concentration of 40%, stirred at 40℃ for 30 minutes, then diluted to an ethanol concentration of 20% by adding water, then hydroxypropyl-β-cyclodextrin is added and stirred at 42℃ for 1.8 hours, then rotary evaporation is performed to remove ethanol, wherein the amount of hydroxypropyl-β-cyclodextrin used is 1:1.1 of the total amount of baicalein and asiaticoside, and the weight / volume ratio of the total amount of baicalein and asiaticoside and the initial 40% ethanol aqueous solution is 1 g:10 ml;

[0088] Step 2, modified trehalose and hydroxypropyl-β-cyclodextrin are dissolved in a 5% ethanol aqueous solution at 50℃, the weight ratio of modified trehalose and the ethanol aqueous solution is 1:20, then polyethylene glycol phospholipid, L-proline and modified mannitol are sequentially added, and mixed at 25℃;

[0089] Step 3, nano-silicon dioxide is added to the freeze-drying protective agent, ultrasonic dispersion is performed (200W, 15 minutes), then the pretreated VEGF, copper peptide and acetyl tetrapeptide-3, baicalein and asiaticoside of step 1 are added and mixed evenly; Step 4, the mixed solution obtained in step 3 is kept at 4℃ for 30 minutes, cooled to -80℃ and kept for 2 hours, then dried at -50℃ and 10Pa for 24 hours, and then dried at 20℃ and 1Pa for 12 hours; ground through an 80-mesh sieve and packaged with nitrogen, thereby obtaining the freeze-dried powder composition for hair regeneration.

[0090] Example 4

[0091] Among them, modified trehalose 30 parts, modified mannitol 18 parts, the rest is the same as example 1.

[0092] Example 5

[0093] Among them, modified trehalose 32 parts, modified mannitol 15 parts, the rest is the same as example 1.

[0094] Comparative Example 1

[0095] A freeze-dried powder composition for hair regeneration, the freeze-drying protective agent of which uses ordinary trehalose and mannitol, and the rest is the same as example 1.

[0096] Comparative Example 2

[0097] A lyophilized powder composition for hair regrowth, which uses modified trehalose and ordinary mannitol in the lyophilization protective agent, and the rest is the same as Example 1.

[0098] Comparative Example 3

[0099] A lyophilized powder composition for hair regrowth, which uses ordinary trehalose and modified mannitol in the lyophilization protective agent, and the rest is the same as Example 1.

[0100] Comparative Example 4

[0101] Among them, modified trehalose 37 parts, modified mannitol 9 parts, and the rest is the same as Example 1.

[0102] Comparative Example 5

[0103] Among them, modified trehalose 22 parts, modified mannitol 22 parts, and the rest is the same as Example 1.

[0104] Experimental test:

[0105] Take the lyophilized powder composition of the examples and comparative examples, add water for injection, the ratio of lyophilized powder to water for injection is 1mg / 2ml, shake and dissolve, the appearance and dissolution state of the lyophilized powder are shown in Table 1.

[0106] Take 20 mice (30 C57BL6 mice, 8 weeks old, half male and half female, average weight 25g), 24 hours before the experiment, use a depilatory agent to depilate the drug area (both sides of the back), the depilation range is 2cm x 2cm on both sides, position the left side of the mouse back as the control area, and the right side as the test area, give the control area 0.5mL of normal saline, and the test area is given 0.5mL of a solution prepared by dissolving the lyophilized powder of Example 1 and Comparative Example 1 in normal saline, wherein the ratio of lyophilized powder to normal saline is 1mg:3ml, cover with gauze for protection, and fix with adhesive tape, 24 hours later, remove the test object, wash with warm water, and observe the application site for 1h, 2h, 4h, 8h, 12h for redness and edema, the results show that there is no redness and edema in the test area and the control area, indicating that the lyophilized powder prepared by the application has little skin irritation.

[0107] Animal hair growth promoting experiment:

[0108] The mouse alopecia model was established by shaving the backs of mice with a razor. Sodium sulfide was applied to both sides of the backs of the mice to induce the hair to enter the growth phase. After hair removal, hair growth was induced on the backs of the mice, covering an area of ​​approximately 2cm × 3cm. Then, hair removal cream was applied according to the instructions to remove any remaining hair, confirming that the hair growth of the mice was in the resting phase, thus establishing the mouse alopecia model. Mice were randomly divided into three groups of 10 mice each. Each group was treated once daily with physiological saline (blank control group), the lyophilized powder composition of the examples and comparative examples, respectively. In the examples and comparative examples, a 30% solution of physiological saline was prepared, and 0.2 mL was applied each time for 21 consecutive days. After 21 days of treatment, skin from the hair-removed area on the backs of the mice was taken. A circular skin patch was taken from the same location in the hair-removed area of ​​each mouse using a 20mm punch. All body hair on the skin patch was scraped off with a scalpel, and the weight was measured using an analytical balance. The mean weight of the hair in each group was calculated, and the results are shown in Table 1 below.

[0109] Table 1. Test Results

[0110]

[0111]

[0112] As shown in the table above, by using the technical solution of the present invention, and simultaneously adding modified trehalose and modified mannitol to the freeze-drying protectant, the resulting freeze-dried powder composition has a fine and uniform appearance, fast reconstitution time, and good solubility. In contrast, the freeze-dried powder compositions obtained in Comparative Examples 1-3 have clumps, longer reconstitution time, and turbid solutions with precipitation. Furthermore, the hair weight of Examples 1-3 of the present invention is also better than that of Comparative Examples 1-3. The results indicate that the freeze-dried powder obtained by using the freeze-drying protectant of modified trehalose and modified mannitol of the present invention significantly improves its uniformity and solubility, and enhances the stability and bioactivity of the active ingredients.

[0113] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0114] 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, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A lyophilized powder composition for hair regrowth, characterized by, The following components are included by weight parts: vascular endothelial growth factor 5-15 parts, platelet-derived growth factor 3-10 parts, copper peptide 1-5 parts, acetyl tetrapeptide-3 0.5-3 parts, baicalein 2-8 parts, asiaticoside 1-5 parts, freeze-drying protective agent 20-40 parts, nano-silicon dioxide 1-3 parts; The freeze-drying protective agent includes the following components by weight parts: modified trehalose 25-35 parts, modified mannitol 10-20 parts, hydroxypropyl-β-cyclodextrin 15-25 parts, pegylated phospholipid 5-8 parts, L-proline 8-10 parts; The preparation method of the modified trehalose comprises the following steps: Step a: trehalose and sodium tripolyphosphate are reacted at pH 9.4-9.6 and 50-55°C for 4-5 hours, then purified by ion exchange after dialysis to obtain product 1; Step b: product 1 and palmitoyl chloride are reacted in argon-protected tetrahydrofuran at 26-30°C for 2-3 hours, and then purified by supercritical CO2 extraction to obtain product 2; Step c: 100 parts by weight of product 2 and 220-260 parts by weight of mPEG-NHS are reacted in PBS at pH 7.2-7.6 at 2-8°C for 10-14 hours, then filtered by tangential flow and freeze-dried to obtain the product. The preparation method of the modified mannitol is as follows: comprising the following steps: Step A: mannitol and succinic anhydride are mixed, 4-dimethylaminopyridine and a solvent are added, and the mixture is reacted at 50-55°C under nitrogen atmosphere for 5-6 hours; Step B: the reaction solution is distilled under reduced pressure to remove the solvent, precipitated with ether, and then purified by ultrafiltration; Step C: spray drying, inlet temperature 70-75°C, outlet temperature 50-55°C.

2. The lyophilized powder composition for hair re growth according to claim 1, characterized in that, In step a, the weight ratio of trehalose to sodium tripolyphosphate is 100:58-75.

3. The lyophilized powder composition for hair re growth according to claim 1, characterized in that, In step b, the weight ratio of product 1 to palmitoyl chloride and tetrahydrofuran is 100:45-50:300-500.

4. The lyophilized powder composition for hair re-growth as claimed in claim 1, wherein, In step A, the molar ratio of mannitol to succinic anhydride and 4-dimethylaminopyridine is 1:0.25-0.35:0.03-0.08, the solvent is DMF or DMSO, and the amount used is 50-100 mL of DMF or DMSO per mole of mannitol.

5. The method for preparing a lyophilized powder composition for hair re¬ growth according to any one of claims 1 to 4, characterized in that The method comprises pretreating vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside; mixing modified trehalose, hydroxypropyl-β-cyclodextrin, pegylated phospholipid, L-proline, and modified mannitol to prepare a freeze-drying protective agent; and then mixing the pretreated vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside with the freeze-drying protective agent and nano-silicon dioxide, and freeze-drying to obtain the product.

6. The method for preparing a lyophilized powder composition for hair re growth according to claim 5, wherein The pretreatment comprises dissolving vascular endothelial growth factor and platelet-derived growth factor in PBS buffer containing 0.1% human serum albumin, and storing the mixture at 2-8°C in the dark, wherein the weight volume ratio of the total mass of vascular endothelial growth factor and platelet-derived growth factor to PBS buffer is 1 mg:10-12 ml.

7. The method for preparing a lyophilized powder composition for hair re growth according to claim 5, wherein The pretreatment comprises dissolving copper peptide and acetyl tetrapeptide-3 in an aqueous solution containing 1-2% propylene glycol, 0.05% ethylenediaminetetraacetic acid and 0.05% Tween 80, and the weight / volume ratio of the total weight of copper peptide and acetyl tetrapeptide-3 to the aqueous ethylenediaminetetraacetic acid solution is 1 mg: 1-2 ml.

8. The method for preparing a lyophilized powder composition for hair re growth according to claim 5, wherein The pretreatment comprises adding baicalein and asiaticoside to an aqueous solution of hydroxypropyl-beta-cyclodextrin and stirring the reaction at 38-42 DEG C for 1.5-2.5 hours, wherein the amount of hydroxypropyl-beta-cyclodextrin used is 1:0.9-1.1 of the total amount of baicalein and asiaticoside.

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