Freeze-dried powder composition for hair regeneration and preparation method thereof

Abstract

The invention 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. The freeze-dried powder composition comprises 5-15 parts of a vascular endothelial growth factor, 3-10 parts of a 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 a freeze-drying protective agent and 1-3 parts of nano silicon dioxide. The preparation method comprises the following steps: mixing the pretreated vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein and asiaticoside with a freeze-drying protective additive and nano silicon dioxide, and freeze-drying. The freeze-dried powder composition can promote hair growth, improve scalp microenvironment and provide lasting support for healthy growth of hair. According to the preparation method, the stability and biological activity of the active ingredients are improved, and the overall performance and safety of the product are improved.

Description

Technical Field

[0001] The present invention 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. Background Art

[0002] In recent years, the number of people suffering from hair loss has been increasing due to a variety of factors, including environmental factors, work pressure, and genetics. Hair loss is caused by many internal factors, such as aging and hormonal imbalances, as well as various diseases and severe burns or wounds. It is often accompanied by thinning of the skin in the bald areas, which is caused by the loss of follicular function.

[0003] Currently, several treatment options for hair loss include oral or topical medications, hair transplantation, and more. However, these treatments have limitations, with medications providing only temporary relief. For example, oral finasteride has sexual side effects and can cause birth defects, and discontinuation of medication can accelerate hair loss. Hair transplantation is a reliable surgical treatment, but it has a low transplant survival rate, high costs, and certain risks.

[0004] With the development of society, people pay more and more attention to hair, which has both protective and aesthetic functions. The study of hair follicle regeneration has practical and important significance.

[0005] Hair regeneration has been a key research area in medical aesthetics and skin health management in recent years. With increasing demand for health and beauty, the development of effective and safe hair regeneration products has become a research hotspot. However, the hair regeneration process is complex, involving the synergistic effects of multiple bioactive factors, which play a vital role in promoting hair follicle growth, repairing damaged 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. By using low-temperature drying, freeze-drying technology avoids the damage of active ingredients caused by high temperatures, while also improving product stability and shelf life. However, while traditional lyoprotectants such as trehalose and mannitol can protect bioactive substances to a certain extent, their effectiveness is limited under specific conditions and may affect the bioavailability of the final product.

[0007] Despite the progress made in existing technologies, several challenges remain. For example, how to further improve the stability and bioavailability of bioactive substances and how to optimize the formulation of lyoprotectants to accommodate different types of bioactive substances. Addressing these issues will contribute to the development of more effective and safer hair regeneration products, address market needs, and promote the advancement of related technologies. Summary of the Invention

[0008] To solve the above problems, the present invention provides a freeze-dried powder composition for hair regeneration and a preparation method thereof. The freeze-dried powder composition has good stability, achieves efficient hair regeneration, and significantly improves scalp health. It can not only promote hair growth, but also fundamentally improve the scalp microenvironment, providing lasting support for the healthy growth of hair. Through the pretreatment process, the preparation method not only improves the stability and biological activity of the active ingredients, but also optimizes the uniformity and solubility of the composition, thereby enhancing the overall performance and safety of the product. During the freeze-drying process, multi-stage stepped heating is adopted, which can make the water sublimate evenly and avoid the problem of uneven local crystallization.

[0009] The technical solution adopted by the present invention to achieve the above object is as follows:

[0010] A freeze-dried powder composition for hair regeneration, comprising the following components in parts by weight:

[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-3, 2-8 parts of baicalein, 1-5 parts of asiaticoside, 20-40 parts of freeze-drying protectant, 1-3 parts of nano-silica;

[0012] The freeze-drying protectant comprises the following components in parts by weight: 25-35 parts of modified trehalose, 10-20 parts of modified mannitol, 15-25 parts of hydroxypropyl-β-cyclodextrin, 5-8 parts of polyethylene glycolated phospholipid, 8-10 parts of L-proline;

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

[0014] Step a, reacting trehalose with sodium tripolyphosphate at pH 9.4-9.6 and 50-55 °C for 4-5 hours, followed by dialysis and ion exchange purification to obtain product 1;

[0015] Step b, reacting product 1 with palmitoyl chloride in argon-protected tetrahydrofuran at 26-30 °C for 2-3 hours, followed by supercritical CO2 extraction purification to obtain product 2;

[0016] Step c, reacting 100 parts by weight of product 2 with 220-260 parts by weight of mPEG-NHS in PBS at pH 7.2-7.6 at 2-8 °C for 10-14 hours, followed by tangential flow filtration and freeze-drying 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 produce phosphorylated trehalose. The introduction of phosphate groups increases the negative charge density of trehalose, giving it better water solubility and ion exchange ability. In step b, the unreacted hydroxyl groups (-OH) of trehalose react with palmitoyl chloride (C 15 H 31 COCl) to undergo an esterification reaction, forming palmitate bonds. The introduction of palmitate bonds changes the molecular structure of trehalose, transforming it from a hydrophilic molecule into an amphiphilic molecule with hydrophobic properties. The hydrophobic end (palmitoyl chain) of the amphiphilic modified trehalose adsorbs on the surface of ice crystals, and the hydrophilic end (phosphorylated / PEGylated part) binds to water molecules, forming a "molecular shield": The modified trehalose restricts the ice crystal particle size through steric hindrance, reduces mechanical damage to proteins, and improves the activity retention rate of growth factors. The hydrophobic chain binds to the hydrophobic region of proteins, preventing protein-water interface denaturation during lyophilization. The hydrophobic chain can also encapsulate 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 interactions, increasing the glass transition temperature, avoiding the collapse of the amorphous phase during lyophilization, enhancing mechanical strength, and preventing the cracking of the lyophilized powder. At the same time, the palmitoyl chain inserts into the hydrophobic cavity of cyclodextrin, forming a "supramolecular carrier" to improve the solubility of baicalein. The amphiphilic modified trehalose is oriented at the solid-liquid interface during reconstitution, reducing the surface tension and shortening the reconstitution time. By reacting with mPEG-NHS (methoxypolyethylene glycol-succinimide ester), a polyethylene glycol chain (PEG chain) is further introduced onto the trehalose molecule. The PEG chain has good biocompatibility and water solubility, which can further improve the stability and dispersibility of trehalose, effectively prevent the aggregation of growth factors, and help to uniformly encapsulate and protect other active ingredients.

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

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

[0023] Step B, remove the solvent from the reaction solution by vacuum distillation, and perform ultrafiltration purification after precipitation with ether;

[0024] Step C, perform spray drying, with 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, succinic anhydride, and 4-dimethylaminopyridine is 1:0.25 - 0.35:0.03 - 0.08, the solvent is DMF or DMSO, and the dosage is 50 - 100 mL of DMF or DMSO per mole of mannitol.

[0026] By reacting with succinic anhydride, new functional groups (such as carboxyl groups) are introduced into the molecular structure of mannitol, enhancing the molecular polarity, improving water solubility, reducing crystallinity, forming an amorphous structure, and avoiding "caking" during the reconstitution of the freeze-dried powder. This modified mannitol can dissolve and disperse better in the freeze-drying 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 freeze-drying damage. In synergy with L-proline and hydroxypropyl-β-cyclodextrin, it enhances the stability of the glassy matrix. In synergy with modified trehalose, the carboxylic acid group forms an electrostatic complex with phosphorylated trehalose, optimizing the porosity of the freeze-dried powder and increasing the specific surface area. When combined with nano-silica, the carboxylic acid group anchors to the surface of silica through hydrogen bonds, enhancing drug dispersion. The carboxyl group can also improve the compatibility with the skin cutin layer and promote hair follicle penetration.

[0027] The preparation method of the above-mentioned freeze-dried powder composition for hair regeneration includes pre-treating vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside; mixing modified trehalose, hydroxypropyl-β-cyclodextrin, polyethylene glycolated phospholipid, L-proline, and modified mannitol to obtain a freeze-drying protectant; then mixing the pre-treated vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside with the freeze-drying protectant and nano-silica, and freeze-drying to obtain the product.

[0028] Preferably, the pre-treatment includes dissolving vascular endothelial growth factor and platelet-derived growth factor in PBS buffer containing 0.1% human serum albumin and storing them in the dark at 2 - 8°C. Among them, the total mass of vascular endothelial growth factor and platelet-derived growth factor and the weight-volume ratio of 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. PBS buffer (pH 7.4) provides a stable ionic environment to avoid protein denaturation. HSA synergizes with the subsequent added freeze-drying protectant (such as modified trehalose) to form a "protein-sugar" complex, reducing ice crystal damage.

[0030] Preferably, the pretreatment includes 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. The weight-volume ratio of the total weight of copper peptide and acetyl tetrapeptide-3 to the aqueous solution of ethylenediaminetetraacetic acid is 1 mg: 1-2 ml.

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

[0032] Preferably, the pretreatment includes adding baicalein, asiaticoside and hydroxypropyl-β-cyclodextrin to an aqueous ethanol solution and stirring and reacting at 38-42 °C for 1.5-2.5 hours. Among them, the dosage of hydroxypropyl-β-cyclodextrin and the total amount of baicalein and asiaticoside are 1: 0.9-1.1, 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 compounds with baicalein and asiaticoside, significantly improving their solubility in water. The formation of inclusion compounds can protect baicalein and asiaticoside from oxidation and degradation and improve their stability during freeze-drying and storage. The formation of inclusion compounds helps to improve the bioavailability of baicalein and asiaticoside, enabling them to play a more effective role when used.

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

[0035] The lyophilized powder composition of the present invention contains a variety of key growth factors (such as vascular endothelial growth factor, platelet-derived growth factor) and bioactive peptides (such as copper peptide, acetyl tetrapeptide-3). These components can act synergistically to promote the proliferation and differentiation of hair follicle cells, induce the transformation of hair follicles from the resting phase to the growth phase, and thus significantly improve the hair regeneration ability. Natural plant components such as baicalein and asiaticoside have antioxidant and anti-inflammatory effects, can improve the scalp microenvironment, reduce the inflammatory reaction around hair follicles, and create good conditions for hair growth. Components such as modified trehalose, modified mannitol, hydroxypropyl-β-cyclodextrin, polyethylene glycolated phospholipid and L-proline in the lyophilization protectant can form a stable glassy structure, prevent the denaturation and inactivation of active ingredients during lyophilization and storage, and significantly extend the product's shelf life. Nano-silica can adsorb on the surface of active ingredients, prevent their aggregation and precipitation, and further improve the stability and solubility of the lyophilized powder. This lyophilized powder composition has good stability, realizes efficient hair regeneration, and significantly improves scalp health. It can not only promote hair growth, but also fundamentally improve the scalp microenvironment and provide lasting support for the healthy growth of hair.

[0036] The lyophilized powder composition for hair regeneration provided by the present invention combines two growth factors. Among them, vascular endothelial growth factor promotes angiogenesis around hair follicles, improves the blood supply to the dermal papilla, and provides sufficient nutrition for hair follicles; platelet-derived growth factor can directly stimulate the activation of hair follicle stem cells, activate the proliferation of hair follicle stem cells, and promote hair growth; the two act synergistically to extend the growth phase of hair follicles, promote the proliferation and differentiation of hair follicle cells, and induce the transformation of hair follicles from the resting phase to the growth phase. In the polypeptide synergistic system, copper peptide provides a good environment for the healthy growth of hair by promoting hair follicle growth, antioxidant and anti-inflammatory effects, and repairing damaged tissues; while acetyl tetrapeptide-3 further improves the scalp condition and promotes hair growth through anti-inflammatory and skin-tightening effects. The combination of the two can more comprehensively enhance the effect of hair regeneration. Natural ingredients such as baicalein and asiaticoside have antioxidant and anti-inflammatory effects, can improve the scalp environment, and reduce the inflammatory reaction around hair follicles, thus creating good conditions for hair growth.

[0037] Trehalose is a natural non-reducing disaccharide with good stability. Modified trehalose is triple-modified by introducing phosphate groups, palmitoyl groups, and PEG chains, achieving the synergistic effects of charge stability, interfacial activity, and steric protection, significantly enhancing its performance as a lyoprotectant. Modified trehalose not only improves its own chemical and thermal stability but also enhances its solubility and dispersibility, while strengthening the protection ability for other active ingredients. These properties enable modified trehalose to play an important protective and synergistic role in the hair regeneration lyophilized powder composition. The phosphate group forms hydrogen bonds with the hydroxyl groups of cyclodextrin, enhancing the inclusion stability of baicalein, etc. It jointly constructs a protective layer with L-proline to reduce protein dehydration damage. During the lyophilization process, modified trehalose can form a stable glassy structure to prevent the denaturation and inactivation of active ingredients. Components such as hydroxypropyl-β-cyclodextrin and polyethylene glycolated phospholipids in the lyoprotectant can form inclusion compounds or complexes with active ingredients such as growth factors, further improving their stability. This synergistic protection enables the lyophilized powder to maintain high biological activity during storage and transportation. Nanoscale silica has good dispersibility and adsorbability, and can adsorb on the surface of active ingredients to prevent their aggregation and precipitation. This not only improves the solubility of the lyophilized powder but also extends its shelf life.

[0038] Modified mannitol introduces new functional groups through specific chemical reactions, significantly improving its solubility, stability, and biocompatibility. In the lyoprotectant, modified mannitol can better protect other active ingredients, enhancing the overall performance and safety of the product.

[0039] In the preparation method of the present invention, the pretreatment process not only improves the stability and biological activity of the active ingredients but also optimizes the uniformity and solubility of the composition, thereby enhancing the overall performance and safety of the product. In addition, the pretreatment process simplifies the production process, improving production efficiency and repeatability. Adopting a multi-stage stepped temperature rise during the lyophilization process can enable the uniform sublimation of water, avoiding the problem of non-uniform local crystallization. This process can improve the uniformity and stability of the lyophilized powder. Detailed implementation manners

[0040] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0041] Vascular endothelial growth factor brands: Yuanye; Platelet-derived growth factor - recombinant human platelet-derived growth factor BB (PDGF-BB), brand, Abbkine; Copper peptide, Hubei Xinyuhong Biomedical Technology Co., Ltd.; Acetyl tetrapeptide-3, Xi'an Shilin Biotechnology Co., Ltd.; EDTA, Beijing Aomijia De Pharmaceutical Technology Co., Ltd.; Baicalein, with an active ingredient content of 98%, Yangling Ciyuan Biotechnology Co., Ltd.; Asiaticoside, 80 mesh, Lanzhou Wateles Biotechnology Co., Ltd.; Hydroxypropyl-β-cyclodextrin, Hubei Yamade Biomedical Co., Ltd.; Nano-silica, 50 - 80 nm; Trehalose, Zhejiang Binmei Biotechnology Co., Ltd.; Mannitol, Xi'an Hongyao Pharmaceutical Excipients Co., Ltd.; L-proline, Anhui Zhonghong Bioengineering Co., Ltd.; Sodium tripolyphosphate, Hunan Xunao Bioengineering Co., Ltd.; Palmitoyl chloride, Kangdis Chemical Industry; PBS buffer, Shanghai Zeye Biotechnology Co., Ltd.; Human serum albumin, brand: Targetmol; Polyethylene glycolated phospholipid, DSPE-PEG2000, purity 95%, Shaanxi Xingbei Aike Biotechnology Co., Ltd.; MPEG-NHS (methoxypolyethylene glycol active ester) with a molecular weight of 2000, Shanghai Yuanye Biotechnology Co., Ltd.; Succinic anhydride, Wuhan Simac Biotechnology Co., Ltd. The raw materials used in the following examples are all ordinary commercially available products.

[0042] Example 1

[0043] A freeze-dried powder composition for hair regeneration, comprising the following components in parts by weight:

[0044] 10 parts of vascular endothelial growth factor, 6 parts of platelet-derived growth factor, 3 parts of copper peptide, 2 parts of acetyl tetrapeptide-3, 5 parts of baicalein, 3 parts of asiaticoside, 30 parts of freeze-drying protectant, and 2 parts of nano-silica;

[0045] The freeze-drying protectant comprises the following components in parts by weight: 28 parts of modified trehalose, 15 parts of modified mannitol, 20 parts of hydroxypropyl-β-cyclodextrin, 6 parts of polyethylene glycolated phospholipid, and 9 parts of L-proline;

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

[0047] Step a: React 100 parts by weight of trehalose with 65 parts by weight of sodium tripolyphosphate at pH 9.4 - 9.6 and 53 °C for 4.5 hours. Adjust the pH to 7.0 ± 0.2 with 1M HCl to terminate the reaction. Perform two-stage dialysis successively using a dialysis bag with a molecular weight cut-off of 3.5 kDa: First stage: Dialyze with 0.1M NaCl solution at a volume ratio of 1:20 at 4 °C for 4 hours; Second stage: Dialyze with ultrapure water at a volume ratio of 1:50 at 4 °C for 24 hours. Load the dialysis solution onto a DEAE cellulose column and elute with a linear gradient of 0 - 0.5M NaCl at a flow rate of 1 mL / min. Collect the electroelution fractions, concentrate by rotary evaporation to a solid content of ≥ 20%, and freeze-dry to obtain Product 1;

[0048] Step b: React 100 parts by weight of Product 1 with 47 parts by weight of palmitoyl chloride in 400 parts of tetrahydrofuran under argon protection at 28 °C for 2.5 hours. Drop the reaction solution into 10 times the volume of methanol at -20 °C for precipitation, centrifuge at 8000 rpm for 15 minutes, and collect the precipitate. Purify by supercritical CO2 extraction under the following conditions: pressure 80 bar, temperature 35 °C; CO2 flow rate 8 L / min; extract cyclically 3 times, 30 minutes each time; dry in vacuum at 40 °C for 24 hours with a vacuum degree of ≤ 5 Pa to obtain Product 2;

[0049] Step c: React 100 parts by weight of Product 2 with 240 parts by weight of mPEG-NHS in PBS at pH 7.2 - 7.6 at 5 °C for 12 hours. Purify using a 10 kDa tangential flow filtration system, and then freeze-dry. The freeze-drying process is as follows: pre-freeze at -80 °C for 2 hours, dry at -40 °C / 15 Pa for 20 hours, and perform secondary drying at 25 °C / 5 Pa for 6 hours to obtain the product.

[0050] The preparation method of the modified mannitol is as follows: It includes the following steps:

[0051] Step A: Add mannitol, succinic anhydride, and 4-dimethylaminopyridine to DMF, and react under a nitrogen atmosphere at 52 °C for 5.5 hours. The molar ratio of mannitol, succinic anhydride, and 4-dimethylaminopyridine is 1:0.3:0.05, and the amount of DMF used is 75 mL of DMF per mole of mannitol;

[0052] Step B: Remove the solvent from the reaction solution by vacuum distillation, precipitate with ether, and then purify by ultrafiltration;

[0053] Step C: Perform spray drying with an inlet temperature of 72 °C and an outlet temperature of 52 °C to obtain the product.

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

[0055] Step 1, Pretreatment: Vascular endothelial growth factor and platelet-derived growth factor are dissolved in PBS buffer (pH 7.2 - 7.6) containing 0.1% human serum albumin (HSA) and stored in the dark at 6°C. The total mass of vascular endothelial growth factor and platelet-derived growth factor to the weight / volume ratio of PBS buffer is 1 mg: 11 ml; Copper peptide and acetyl tetrapeptide-3 are dissolved in an aqueous solution containing 1.5% propylene glycol, 0.05% EDTA (ethylenediaminetetraacetic acid) and 0.05% Tween 80. The weight / volume ratio of copper peptide and acetyl tetrapeptide-3 to the EDTA aqueous solution is 1 mg: 1 ml; Baicalein and asiaticoside are added to an aqueous ethanol solution with an ethanol concentration of 40% and stirred at 40°C for 30 minutes, then diluted with water to an ethanol concentration of 20%, and hydroxypropyl-β-cyclodextrin is added and stirred at 40°C for 2 hours, and then ethanol is removed by rotary evaporation. Among them, the dosage of hydroxypropyl-β-cyclodextrin to the total amount of baicalein and asiaticoside is 1:1, and the total amount of baicalein and asiaticoside to the weight / volume ratio of the initial 40% aqueous ethanol solution is 1 g: 12 ml;

[0056] Step 2, Dissolve modified trehalose and hydroxypropyl-β-cyclodextrin in a 5% aqueous ethanol solution at 45°C. The weight ratio of modified trehalose to the aqueous ethanol solution is 1:18, and then polyethylene glycolated phospholipid, L-proline and modified mannitol are added in sequence and stirred and mixed at 30°C to obtain a lyoprotectant;

[0057] Step 3, Add nano-silica to the lyoprotectant, after ultrasonic dispersion (150 W, 18 minutes), add the vascular endothelial growth factor, copper peptide and acetyl tetrapeptide-3, baicalein and asiaticoside pretreated in Step 1, and mix well; Step 4, Keep the mixture obtained in Step 3 at 4°C for 30 minutes, cool down to -80°C and keep for 2 hours; then dry at -48°C and 12 Pa for 30 hours, and then dry at 22°C and 3 Pa for 10 hours; pulverize and pass through a 100-mesh sieve, and package with nitrogen filling to obtain the product.

[0058] Example 2

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

[0060] 15 parts of vascular endothelial growth factor, 3 parts of platelet-derived growth factor, 5 parts of copper peptide, 0.5 part of acetyl tetrapeptide-3, 8 parts of baicalein, 1 part of asiaticoside, 40 parts of lyoprotectant, 1 part of nano-silica;

[0061] The lyoprotectant comprises the following components by weight: 35 parts of modified trehalose, 10 parts of modified mannitol, 15 parts of hydroxypropyl-β-cyclodextrin, 8 parts of polyethylene glycolated phospholipid, 8 parts of L-proline;

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

[0063] Step a: React 100 parts by weight of trehalose with 75 parts by weight of sodium tripolyphosphate at pH 9.4 - 9.6 and 55°C for 4 hours, and purify (the same as in Example 1) to obtain Product 1;

[0064] Step b: React 100 parts by weight of Product 1 with 50 parts by weight of palmitoyl chloride in 300 parts of tetrahydrofuran under argon protection at 26°C for 3 hours, and purify by supercritical CO2 extraction (the same as in Example 1) to obtain Product 2;

[0065] Step c: React 100 parts by weight of Product 2 with 260 parts by weight of mPEG - NHS in PBS at pH 7.2 - 7.6 at 2°C for 14 hours, perform tangential flow filtration and then freeze - dry (the same as in Example 1). The freeze - drying process is as follows: pre - freeze at - 80°C for 2 hours, dry at - 40°C / 15 Pa for 20 hours, and perform secondary drying at 25°C / 5 Pa for 6 hours to obtain the product.

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

[0067] Step A: Add mannitol, succinic anhydride, and 4 - dimethylaminopyridine into DMF, and react at 55°C under a nitrogen atmosphere for 5 hours. The molar ratio of mannitol, succinic anhydride, and 4 - dimethylaminopyridine is 1:0.25:0.03, and the amount of DMF used is 50 mL of DMF per mole of mannitol;

[0068] Step B: Remove the solvent from the reaction solution by vacuum distillation, precipitate with ether, and then purify by ultrafiltration;

[0069] Step C: Spray - dry at an inlet temperature of 75°C and an outlet temperature of 55°C to obtain the product.

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

[0071] Step 1, pretreatment: Vascular endothelial growth factor and platelet-derived growth factor are dissolved in PBS buffer (pH 7.2 - 7.6) containing 0.1% human serum albumin (HSA), and stored in the dark at 4°C. The total mass of vascular endothelial growth factor and platelet-derived growth factor and the weight-to-volume ratio of the PBS buffer is 1 mg: 12 ml; Copper peptide and acetyl tetrapeptide-3 are dissolved in an aqueous solution containing 1% propylene glycol, 0.05% EDTA (ethylenediaminetetraacetic acid) and 0.05% Tween 80. The weight-to-volume ratio of copper peptide and acetyl tetrapeptide-3 to the EDTA aqueous solution is 1 mg: 2 ml; Baicalein and asiaticoside are added to an aqueous ethanol solution with an ethanol concentration of 40% and stirred at 40°C for 30 minutes, then diluted with water to an ethanol concentration of 20%, and hydroxypropyl-β-cyclodextrin is added and stirred at 38°C for 2.5 hours, and then ethanol is removed by rotary evaporation. Among them, the dosage of hydroxypropyl-β-cyclodextrin and the total amount of baicalein and asiaticoside are 1:0.9, and the total amount of baicalein and asiaticoside and the weight-to-volume ratio of the initial 40% aqueous ethanol solution is 1 g: 15 ml;

[0072] Step 2, Dissolve modified trehalose and hydroxypropyl-β-cyclodextrin in a 5% aqueous ethanol solution at 40°C. The weight ratio of modified trehalose to the aqueous ethanol solution is 1:15, and then polyethylene glycolated phospholipid, L-proline and modified mannitol are added in sequence, and stirred and mixed at 28°C to obtain a lyoprotectant;

[0073] Step 3, Add nano-silica to the lyoprotectant, after ultrasonic dispersion (100 W, 20 minutes), add the vascular endothelial growth factor, copper peptide and acetyl tetrapeptide-3, baicalein and asiaticoside pretreated in Step 1, and mix well; Step 4, Keep the mixture obtained in Step 3 at 4°C for 30 minutes, cool down to -80°C and keep it for 2 hours; then dry at -45°C and 15 Pa for 36 hours, and then dry at 25°C and 5 Pa for 8 hours; pulverize and pass through a 120-mesh sieve, and package with nitrogen filling to obtain the product.

[0074] Example 3

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

[0076] 5 parts of vascular endothelial growth factor, 10 parts of platelet-derived growth factor, 1 part of copper peptide, 3 parts of acetyl tetrapeptide-3, 2 parts of baicalein, 5 parts of asiaticoside, 20 parts of lyoprotectant, 3 parts of nano-silica;

[0077] The lyoprotectant comprises the following components by weight: 25 parts of modified trehalose, 20 parts of modified mannitol, 25 parts of hydroxypropyl-β-cyclodextrin, 5 parts of polyethylene glycolated phospholipid, 10 parts of L-proline;

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

[0079] Step a: React 100 parts by weight of trehalose with 58 parts by weight of sodium tripolyphosphate at pH 9.4 - 9.6 and 50 °C for 5 hours, then purify (same as Example 1) to obtain Product 1;

[0080] Step b: React 100 parts by weight of Product 1 with 45 parts by weight of palmitoyl chloride in 500 parts of tetrahydrofuran under argon protection at 30 °C for 2 hours, then purify by supercritical CO2 extraction (same as Example 1) to obtain Product 2;

[0081] Step c: React 100 parts by weight of Product 2 with 220 parts by weight of mPEG-NHS in PBS at pH 7.2 - 7.6 at 8 °C for 10 hours, then perform tangential flow filtration and freeze-dry (same as Example 1). The freeze-drying process is as follows: pre-freeze at -80 °C for 2 hours, dry at -40 °C / 15 Pa for 20 hours, and perform secondary drying at 25 °C / 5 Pa for 6 hours to obtain the product.

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

[0083] Step A: Add mannitol, succinic anhydride, and 4-dimethylaminopyridine into DMF, and react at 50 °C under a nitrogen atmosphere for 6 hours. The molar ratio of mannitol, succinic anhydride, and 4-dimethylaminopyridine is 1:0.35:0.08, and the dosage of DMF is 100 mL of DMF per mole of mannitol;

[0084] Step B: Remove the solvent from the reaction solution by vacuum distillation, precipitate with ether, and then purify by ultrafiltration;

[0085] Step C: Spray-dry at an inlet temperature of 70 °C and an outlet temperature of 50 °C to obtain the product.

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

[0087] Step 1, pretreatment: Vascular endothelial growth factor and platelet-derived growth factor are dissolved in PBS buffer (pH 7.2 - 7.6) containing 0.1% human serum albumin (HSA) and stored in the dark at 8°C. The total mass of vascular endothelial growth factor and platelet-derived growth factor and the weight-to-volume ratio of the PBS buffer 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-to-volume ratio of copper peptide and acetyl tetrapeptide-3 to the EDTA aqueous solution is 1 mg: 1.5 ml; Baicalein and asiaticoside are added to an aqueous ethanol solution with an ethanol concentration of 40% and stirred at 40°C for 30 minutes, then diluted with water to an ethanol concentration of 20%, and hydroxypropyl-β-cyclodextrin is added and stirred at 42°C for 1.8 hours, and then ethanol is removed by rotary evaporation. Among them, the dosage of hydroxypropyl-β-cyclodextrin and the total amount of baicalein and asiaticoside are 1:1.1, and the total amount of baicalein and asiaticoside and the weight-to-volume ratio of the initial 40% aqueous ethanol solution is 1 g: 10 ml;

[0088] Step 2, Dissolve modified trehalose and hydroxypropyl-β-cyclodextrin in a 5% aqueous ethanol solution at 50°C. The weight ratio of modified trehalose to the aqueous ethanol solution is 1:20, and then polyethylene glycolated phospholipid, L-proline and modified mannitol are added in sequence and stirred and mixed at 25°C to obtain a lyoprotectant;

[0089] Step 3, Add nano-silica to the lyoprotectant, after ultrasonic dispersion (200 W, 15 minutes), add the vascular endothelial growth factor, copper peptide and acetyl tetrapeptide-3, baicalein and asiaticoside pretreated in Step 1, and mix well; Step 4, Keep the mixture obtained in Step 3 at 4°C for 30 minutes, cool down to -80°C and keep for 2 hours; then dry at -50°C and 10 Pa for 24 hours, and then dry at 20°C and 1 Pa for 12 hours; pulverize and pass through an 80-mesh sieve, and package under nitrogen to obtain the product.

[0090] Example 4

[0091] Among them, there are 30 parts of modified trehalose and 18 parts of modified mannitol, and the rest are the same as in Example 1.

[0092] Example 5

[0093] Among them, there are 32 parts of modified trehalose and 15 parts of modified mannitol, and the rest are the same as in Example 1.

[0094] Comparative Example 1

[0095] A freeze-dried powder composition for hair regeneration, in which ordinary trehalose and mannitol are used in the lyoprotectant, and the rest are the same as in Example 1.

[0096] Comparative Example 2

[0097] A lyophilized powder composition for hair regeneration, in which modified trehalose and ordinary mannitol are used as the lyoprotectant, and the rest is the same as in Example 1.

[0098] Comparative Example 3

[0099] A lyophilized powder composition for hair regeneration, in which ordinary trehalose and modified mannitol are used as the lyoprotectant, and the rest is the same as in Example 1.

[0100] Comparative Example 4

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

[0102] Comparative Example 5

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

[0104] Experimental test:

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

[0106] Take 20 mice (30 8-week-old C57BL6 mice, 15 males and 15 females, with an average body weight of 25 g). 24 hours before the experiment, use a depilatory to depilate the drug application area (both sides of the back). The depilated area is 2 cm × 2 cm on each side. Designate the left side of the mouse back as the control area and the right side as the test area. Administer 0.5 mL of normal saline to the control area and 0.5 mL of the solution obtained by dissolving the lyophilized powder prepared in the example and Comparative Example 1 in normal saline to the test area. Among them, the ratio of lyophilized powder to normal saline is 1 mg: 3 ml. Cover and protect with a gauze and fix with adhesive tape. After 24 hours, wipe off the test substance and wash it with warm water. Observe whether there is erythema and edema at the application site at 1 h, 2 h, 4 h, 8 h, and 12 h. The results show that there is no erythema and edema in both the test area and the control area, indicating that the lyophilized powder prepared by the present invention has little irritation to the skin.

[0107] Animal hair growth promotion experiment:

[0108] Establishment of a mouse alopecia model: Use a razor to shave, apply sodium sulfide to both sides of the mouse's back to induce hair into the growth phase, depilate, and induce the growth of hair on the mouse's back. The area is approximately 2 cm × 3 cm. Then apply depilatory cream according to the instructions to remove the remaining hair, and confirm that the mouse hair growth is in the telogen phase to establish a mouse alopecia model. The mice are randomly divided into three groups, with 10 mice in each group. Each day, physiological saline (blank control group), the freeze-dried powder compositions of the examples and comparative examples are applied once. Among them, the freeze-dried powder compositions of the examples and comparative examples are prepared with physiological saline to a solution concentration of 30%, 0.2 mL each time, and continuously applied for 21 days. After 21 days of administration, take the skin of the depilated area on the mouse's back, use a 20 mm punch to take circular skin pieces at the same position in the depilated area of each mouse, scrape off all body hair on the skin pieces with a scalpel, weigh them with an analytical balance, and calculate the average value of the hair weights of the mice in each group. The results are shown in Table 1 below.

[0109] Table 1. Detection results

[0110]

[0111]

[0112] As can be seen from the above table, by adopting the technical solution of the present invention, in the lyoprotectant, modified trehalose and modified mannitol are added simultaneously. The obtained freeze-dried powder composition has a delicate and uniform appearance, a fast reconstitution time, and a good dissolution state. While the freeze-dried powder compositions obtained in Comparative Examples 1-3 have caking, a longer reconstitution time, and a turbid solution with precipitation. At the same time, the hair weights of Examples 1-3 of the present invention are also better than those of Comparative Examples 1-3. The results show that the freeze-dried powder obtained by using the lyoprotectant of modified trehalose and modified mannitol of the present invention has greatly improved its uniformity and solubility, and improved the stability and biological activity of the active ingredient.

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

[0114] Although the embodiments of the present application have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application. The scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A freeze-dried powder composition for hair regeneration, characterized in that, Comprising the following components 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, lyoprotectant 20 - 40 parts, nano-silica 1 - 3 parts; The lyoprotectant comprises the following components by weight parts: modified trehalose 25 - 35 parts, modified mannitol 10 - 20 parts, hydroxypropyl-β-cyclodextrin 15 - 25 parts, polyethylene glycolated phospholipid 5 - 8 parts, L-proline 8 - 10 parts; The preparation method of the modified trehalose comprises the following steps: Step a, reacting trehalose with sodium tripolyphosphate at pH 9.4 - 9.6 and 50 - 55 °C for 4 - 5 hours, followed by dialysis and ion exchange purification to obtain product 1; Step b, reacting product 1 with palmitoyl chloride in argon-protected tetrahydrofuran at 26 - 30 °C for 2 - 3 hours, followed by supercritical CO2 extraction and purification to obtain product 2; Step c, reacting 100 parts by weight of product 2 with 220 - 260 parts by weight of mPEG-NHS in PBS at pH 7.2 - 7.6 at 2 - 8 °C for 10 - 14 hours, followed by tangential flow filtration and lyophilization to obtain the product.

2. The freeze-dried powder composition for hair regeneration according to claim 1, wherein In step a, the weight ratio of trehalose to sodium tripolyphosphate is 100:58 - 75.

3. The freeze-dried powder composition for hair regeneration according to claim 1, wherein, In step b, the weight ratio of product 1 to palmitoyl chloride and tetrahydrofuran is 100:45 - 50:300 - 500.

4. The freeze-dried powder composition for hair regeneration according to claim 1, wherein, In step c, the weight ratio of product 2 to mPEG-NHS is 100:100 - 150.

5. The freeze-dried powder composition for hair regeneration according to claim 1, wherein The preparation method of the modified mannitol is as follows: comprising the following steps: Step A, mixing mannitol and succinic anhydride, adding 4-dimethylaminopyridine and a solvent, and reacting at 50 - 55 °C under a nitrogen atmosphere for 5 - 6 hours; Step B, distilling off the solvent from the reaction solution under reduced pressure, followed by precipitation with ether and ultrafiltration purification; Step C, spray drying, with an inlet temperature of 70 - 75 °C and an outlet temperature of 50 - 55 °C to obtain the product.

6. The freeze-dried powder composition for hair regeneration according to claim 5, 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, and the solvent is DMF or DMSO, with a dosage of 50 - 100 mL of DMF or DMSO per mole of mannitol.

7. The preparation method of the freeze-dried powder composition for hair regeneration according to any one of claims 1-6, characterized in that, It includes pre-treating vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside; mixing modified trehalose, hydroxypropyl-β-cyclodextrin, polyethylene glycolated phospholipid, L-proline, and modified mannitol to prepare a lyoprotectant; then mixing the pre-treated vascular endothelial growth factor, platelet-derived growth factor, copper peptide, acetyl tetrapeptide-3, baicalein, and asiaticoside with the lyoprotectant and nano-silica, and freeze-drying to obtain the product.

8. The preparation method of the freeze-dried powder composition for hair regeneration according to claim 7, characterized in that, The pre-treatment includes dissolving vascular endothelial growth factor and platelet-derived growth factor in PBS buffer containing 0.1% human serum albumin and storing in the dark at 2 - 8 °C, wherein the total mass of vascular endothelial growth factor and platelet-derived growth factor and the weight / volume ratio of PBS buffer is 1 mg:10 - 12 ml.

9. The preparation method of the freeze-dried powder composition for hair regeneration according to claim 7, wherein The pretreatment includes 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 solution of ethylenediaminetetraacetic acid is 1 mg: 1-2 ml.

10. The preparation method of the freeze-dried powder composition for hair regeneration according to claim 7, characterized in that, The pretreatment includes adding baicalein, asiaticoside and hydroxypropyl-β-cyclodextrin into an aqueous ethanol solution, and stirring and reacting at 38-42 °C for 1.5-2.5 hours. Among them, the dosage of hydroxypropyl-β-cyclodextrin and the total amount of baicalein and asiaticoside are 1: 0.9-1.1.

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