Hair-growing extracting solution and extracting method thereof

Abstract

The invention discloses an extracting solution capable of growing hair and an extracting method thereof, and belongs to the field of hair growing preparations. The extracting solution comprises saw palmetto fruit extract, ginseng root extract, caffeine, a modified cationic liposome compound, modified metal-polyphenol network nanoparticles and a buffer solution. Wherein the modified cationic liposome compound is obtained by forming a lipid membrane from specific phospholipids and cationic lipids, hydrating, ultrasonically preparing a liposome suspension, adding polylysine and targeting peptide, incubating, and performing sucrose density gradient centrifugal purification. The extraction method comprises the following steps: mixing and stirring the components, fixing the volume by using a buffer solution, filtering and sterilizing. According to the extracting solution, modified compounds cooperate with natural components, active substances are delivered to a hair follicle microenvironment in a targeted mode, hair follicle stem cell proliferation related pathways are activated, hair follicle degeneration factors are inhibited, the growth cycle of hair follicles is regulated and controlled from multiple targets, hair growth is effectively promoted, and the obvious hair growing effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of hair growth preparations, and in particular to an extract capable of promoting hair growth and an extraction method thereof. Background Art

[0002] Hair loss has become one of the major challenges affecting human physical and mental health worldwide. Especially in the context of the accelerated pace of modern life and increased environmental pressure, its incidence rate has shown a continuous upward trend. Clinical studies have shown that androgenic alopecia, as the most common type of hair loss, not only affects the patient's appearance, but may also cause psychological problems such as inferiority and anxiety, seriously reducing the quality of life. Although existing treatments such as vasodilators and hormone-regulating drugs have been used clinically for many years, their efficacy is often limited to specific populations and there is a significant risk of side effects. For example, some drugs may cause skin irritation reactions, and long-term use may even lead to systemic adverse reactions such as sexual dysfunction. More importantly, these traditional therapies usually only target a single pathological link (such as the androgen pathway) and are difficult to fully cover the complex network that regulates the hair follicle growth cycle, resulting in limited treatment effects and easy recurrence. Therefore, the development of new, safe and multi-mechanism hair growth preparations has become a research hotspot in the current field of dermatology.

[0003] Natural plant extracts have recently attracted significant attention in hair growth product development due to their widespread availability, safety, and potential for multi-target regulation. Natural ingredients such as ginseng, saw palmetto, and caffeine have been shown to influence hair follicle physiological function through various pathways: some inhibit the activity of enzymes involved in hair follicle miniaturization, thereby slowing hair degeneration; others protect hair follicle cells from environmental damage through antioxidant activity; and still others regulate local microcirculation, providing enhanced nutritional support for hair follicles. However, these natural active ingredients face multiple technical bottlenecks in practical application. First, their molecular structures often result in inefficient transdermal absorption, hindering effective accumulation in the hair follicle microenvironment. Second, the bioavailability of natural extracts is limited by metabolic pathways in the body, and active ingredients may be degraded before reaching their target sites when taken orally or topically. Furthermore, most natural ingredients lack the ability to precisely target hair follicle stem cells or specific signaling pathways, making effective regulation difficult. These issues severely restrict the clinical effectiveness of natural hair growth agents, and technological innovation is urgently needed to overcome these limitations.

[0004] To address these challenges, researchers have attempted to enhance the hair growth potency of natural ingredients through chemical modification or formulation techniques. On the one hand, transdermal permeability of active ingredients has been improved by designing transdermal enhancers or nanocarriers, enabling them to more effectively penetrate the stratum corneum and reach the hair follicle region. On the other hand, structural modifications have been used to impart targeting properties to natural ingredients, enabling them to specifically accumulate in the hair follicle stem cell niche or the inflammatory microenvironment, thereby precisely modulating signaling pathways involved in the hair follicle growth cycle. In recent years, the emergence of novel delivery systems such as liposomes and metal-polyphenol networks has provided new avenues for the targeted delivery of natural active ingredients. These systems achieve active targeting to hair follicles through surface modification while simultaneously extending the duration of action of the active ingredients through sustained release mechanisms within the core. However, existing modification techniques still have numerous drawbacks: some carrier materials lack biocompatibility and may trigger local immune responses; some modification methods lead to decreased stability or loss of active ingredient functionality; and, more importantly, most delivery systems lack the ability to comprehensively regulate the complex pathological mechanisms of the hair follicle microenvironment, making it difficult to simultaneously address multiple issues such as insufficient hair follicle stem cell proliferation, overactivation of inflammatory factors, and hair follicle miniaturization. Therefore, the development of new modified compounds with multiple functional synergies, precise targeting and excellent biocompatibility has become a key breakthrough direction for improving the efficacy of hair growth preparations. Summary of the Invention

[0005] The purpose of the present invention is to provide a hair growth extract and an extraction method thereof, which solve the technical problems of poor targeting and low efficacy of existing preparations.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] A hair growth-promoting extract comprises the following raw materials in parts by weight:

[0008] Saw palmetto fruit extract: 50-120 parts by weight;

[0009] Ginseng root extract: 60-150 parts by weight;

[0010] Caffeine: 10-30 parts by weight;

[0011] Modified cationic liposome complex: 10-30 parts by weight;

[0012] Modified metal-polyphenol network nanoparticles: 5-20 parts by weight;

[0013] Tris-HCl buffer: 765-865 parts by weight;

[0014] The preparation method of the modified cationic liposome complex includes: A1, dissolving dipalmitoylphosphatidylcholine and 1,2-dioleoyl-3-trimethylammoniumpropane in chloroform, evaporating under reduced pressure to form a uniform lipid film, adding a phosphate buffer containing curcumin to hydrate the lipid film, and forming a liposome suspension by probe ultrasound; A2, subsequently adding polylysine and a targeting peptide, incubating at room temperature, and separating and purifying by sucrose density gradient centrifugation.

[0015] In the present invention, the formation process of the modified cationic liposome complex is based on the synergistic effect of the physicochemical properties of phospholipid molecules and targeted modification technology. The core building block of this complex is a composite lipid membrane system composed of dipalmitoylphosphatidylcholine and cationic lipids. The reaction mechanism begins with the ordered self-assembly of lipid molecules in the organic phase. In a chloroform solvent environment, the polar head group and hydrophobic tail of dipalmitoylphosphatidylcholine maintain a dynamic equilibrium through van der Waals forces, forming a stable liquid crystalline structure. Upon addition of the cationic lipid, the two, due to their similar alkyl chain lengths and saturation, spontaneously aggregate through hydrophobic interactions during evaporation under reduced pressure, forming a uniform and dense lipid bilayer film on the container surface. The key to this process lies in the controlled molar ratio of the two lipid components. This specific ratio ensures that the lipid membrane has an appropriate positive charge density and mechanical stability, laying the foundation for subsequent functional modification. The introduction of curcumin-containing phosphate buffer during the hydration stage triggers the rearrangement and reorganization of the lipid membrane. The osmotic pressure of water molecules breaks the surface tension of the lipid membrane, prompting the phospholipid molecules to transform from a two-dimensional planar structure to a three-dimensional closed vesicle. As a hydrophobic active ingredient, curcumin preferentially distributes to the hydrophobic core of the lipid bilayer, where its polar groups form a hydrogen-bonding network with the phospholipid headgroups, achieving stable anchoring at the molecular level. The mechanical energy input from probe ultrasonication causes the lipid bilayer to instantaneously break and restructure, forming a closed liposome structure with a uniform diameter. During this process, the microjets and shock waves generated by cavitation promote rearrangement of lipid molecules, forming a suspension with a multilamellar vesicle structure while ensuring uniform entrapment of curcumin within the liposomes. The introduction of cationic properties imparts a net positive charge to the liposome surface, significantly enhancing its affinity for hair follicle tissue through electrostatic adsorption with negatively charged components on the hair follicle cell membrane (such as heparin sulfate proteoglycans). The targeted modification step achieves functional enhancement through the synergistic effect of polylysine and the targeting peptide. The cationic polymer properties of polylysine further increase the positive charge density on the liposome surface, prolonging its retention time in the hair follicle microenvironment while providing an anchoring site for the targeting peptide. The targeting peptide serves as a specific recognition element, its amino acid sequence designed to precisely match the keratin receptors on the surface of hair follicle stem cells. During room temperature incubation, polylysine and targeting peptides spontaneously assemble on the liposome surface through non-covalent interactions (such as hydrogen bonds and hydrophobic interactions), forming a protective layer with molecular recognition capabilities. Sucrose density gradient centrifugation technology utilizes the separation characteristics of different density media to screen out liposome complexes with specific particle size and charge characteristics through the centrifugal field, ultimately obtaining a functionalized delivery system with uniform purity and activity. This complex achieves efficient delivery of active ingredients such as curcumin to the hair follicle stem cell niche through positive charge-mediated endocytosis and specific uptake guided by the targeting peptide, thereby activating the Wnt / β-catenin signaling pathway to regulate the proliferation and differentiation of hair follicle stem cells.

[0016] According to a preferred embodiment of the present invention, the saw palmetto fruit extract was purchased from Xi'an Tianben Bioengineering Co., Ltd., model TB-SPF-01 (supercritical CO2 extraction, pressure 25 MPa, temperature 40°C, time 1.5 hours, yield ≥8%).

[0017] According to a preferred embodiment of the present invention, the ginseng root extract was purchased from Shaanxi Senfu Natural Products Co., Ltd., model SR-RRT-02 (five-year-old ginseng root, 70% ethanol reflux extraction, solid-liquid ratio 1:10, 65°C extraction twice, each time for 2.5 hours).

[0018] According to a preferred embodiment of the present invention, the caffeine was purchased from Sinopharm Chemical Reagent Co., Ltd., model number C10053658 (purity ≥99%, AR grade).

[0019] According to a preferred embodiment of the present invention, the dipalmitoylphosphatidylcholine was purchased from Shanghai AVT Pharmaceutical Technology Co., Ltd., model AVT-DMPC-001 (purity ≥99%, pharmaceutical grade).

[0020] According to a preferred embodiment of the present invention, the 1,2-dioleoyl-3-trimethylammoniopropane was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., model A101372 (purity ≥98%, DOTAP).

[0021] According to a preferred embodiment of the present invention, the chloroform was purchased from Sinopharm Chemical Reagent Co., Ltd., model number C10017968 (analytical grade, AR grade).

[0022] According to a preferred embodiment of the present invention, the curcumin-containing phosphate buffer solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model number YY12345 (curcumin purity ≥95%, PBS buffer solution pH 7.4).

[0023] According to a preferred embodiment of the present invention, the polylysine was purchased from Beijing Solebow Technology Co., Ltd., model number SL0016 (molecular weight 10 kDa, purity ≥98%).

[0024] According to a preferred embodiment of the present invention, the targeting peptide was purchased from Shanghai Jier Biochemical Co., Ltd., model number JL-KRT75-1 (solid phase synthesis, purity ≥95%, sequence derived from the hair follicle keratin KRT75 specific fragment).

[0025] According to a preferred embodiment of the present invention, the sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd., model number C10021791 (analytical grade, AR grade).

[0026] According to a preferred embodiment of the present invention, the Tris-HCl buffer was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., and the model number was DD-TRIS-6.5 (0.05 M, pH 6.4-6.6, biological buffer).

[0027] According to a preferred embodiment of the present invention, in step A1, the molar ratio of dipalmitoylphosphatidylcholine to 1,2-dioleoyl-3-trimethylammoniopropane is 7:3; and the power of the probe ultrasound is 200-210W.

[0028] According to a preferred embodiment of the present invention, in step A2, the incubation time at room temperature is 2-4 hours.

[0029] According to a preferred embodiment of the present invention, the preparation method of the modified metal-polyphenol network nanoparticles includes: B1, dispersing nano zinc oxide in deionized water, ultrasonically treating to form a uniform dispersion, adding epigallocatechin gallate and ellagic acid, and stirring at room temperature to react to form a metal-polyphenol network structure; B2, subsequently adding hyaluronic acid oligosaccharide to continue the reaction, collecting the retentate by ultrafiltration and freeze-drying.

[0030] In the present invention, the construction of modified metal-polyphenol network nanoparticles is based on the cross-integration of metal-ligand coordination chemistry and nanomaterial surface engineering technology. The process begins with the dispersion of nano-zinc oxide in deionized water. The abundant hydroxyl groups (-OH) on its surface serve as coordination active sites, providing an anchoring basis for the subsequent loading of polyphenol molecules. The cavitation effect generated by ultrasonic treatment causes the zinc oxide particles to be evenly dispersed in the aqueous phase system, avoiding agglomeration while exposing more surface active sites. Epigallocatechin gallate and ellagic acid are natural polyphenol compounds. The multiple phenolic hydroxyl groups (-OH) in their molecular structures interact strongly with the metal ions on the zinc oxide surface through coordination bonds, forming a stable metal-polyphenol network structure. This network is further strengthened by the π-π stacking effect and hydrogen bond network between polyphenol molecules, constructing a protective shell with a three-dimensional structure, effectively encapsulating the nano-zinc oxide core and enhancing its chemical stability. During the stirring reaction, the coordination reaction between the polyphenol molecules and zinc oxide follows a kinetically controlled mechanism. The phenolic hydroxyl oxygen atoms of the polyphenols act as electron donors, forming coordination bonds with zinc ions on the zinc oxide surface. Simultaneously, the hydrogen bond network between the polyphenol molecules promotes the self-assembly of a three-dimensional network structure. This reaction occurs under mild conditions, avoiding the destruction of the active ingredients by high temperatures or strong acids, ensuring that the biological activities of epigallocatechin gallate and ellagic acid are retained. The introduction of hyaluronic acid oligosaccharides further functionalizes the nanoparticles through surface modification techniques. The carboxyl and hydroxyl groups in the hyaluronic acid oligosaccharide molecules interact non-covalently with the active sites surrounding the polyphenol network, forming an outer protective shell. This modification not only imparts the nanoparticles with the ability to target hair follicle tissue (via endocytosis mediated by the hyaluronic acid receptor CD44) but also prevents the premature release of the polyphenol molecules through steric hindrance, achieving controlled release of the active ingredients within the hair follicle microenvironment. Ultrafiltration and freeze-drying steps complete the purification and stabilization of the nanoparticles. The ultrafiltration process utilizes membrane materials with precisely controlled molecular weight cutoffs to selectively remove free polyphenol molecules and small molecule impurities that do not participate in the coordination reaction, retaining the metal-polyphenol network complex within a specific particle size range. Freeze-drying technology maintains the structural integrity of the nanoparticles through low-temperature dehydration, preventing polyphenol oxidation or network collapse caused by high-temperature drying. The resulting modified metal-polyphenol network nanoparticles function through multiple mechanisms: the nano-zinc oxide core provides broad-spectrum antimicrobial properties, inhibiting the proliferation of pathogenic microorganisms in the hair follicle region; epigallocatechin gallate and ellagic acid scavenge free radicals through antioxidant effects, alleviating oxidative stress damage to hair follicle cells; and hyaluronic acid oligosaccharide-mediated targeted delivery allows the active ingredients to be concentrated around the hair follicles, synergistically inhibiting dihydrotestosterone-induced hair follicle miniaturization and inflammatory responses, thereby maintaining the homeostatic balance of the hair follicle microenvironment.

[0031] According to a preferred embodiment of the present invention, the nano zinc oxide is purchased from Nanjing EP Nanomaterials Co., Ltd., with the model number being EP-ZnO-50 (particle size of about 50 nm, purity ≥99%).

[0032] According to a preferred embodiment of the present invention, the epigallocatechin gallate was purchased from Xi'an Tianfeng Biotechnology Co., Ltd., model TF-EGCG-95 (purity ≥95%, active ingredient of green tea polyphenols).

[0033] According to a preferred embodiment of the present invention, the ellagic acid is purchased from Shaanxi Ciyuan Biotechnology Co., Ltd., with the model number being CY-Ellagic-98 (purity ≥ 98%, a natural polyphenol compound).

[0034] According to a preferred embodiment of the present invention, the hyaluronic acid oligosaccharide was purchased from Shanghai Zhaowei Technology Development Co., Ltd., model number ZW-HA4-01 (molecular weight 4 kDa, hyaluronidase-degradable oligosaccharide).

[0035] According to a preferred embodiment of the present invention, the microporous filter membrane was purchased from Tianjin Jinteng Experimental Equipment Co., Ltd., model JT-0.22CM (pore size 0.22 μm, polyethersulfone material, used for sterilization filtration).

[0036] According to a preferred embodiment of the present invention, in step B1, the ultrasonic treatment time is 30-40 min; the molar ratio of ZnO: epigallocatechin gallate: ellagic acid is 1:5:3; and the stirring reaction time is 4-6 h.

[0037] According to a preferred embodiment of the present invention, in step B2, the reaction is continued for 2-4 hours; and the molecular weight cut-off of ultrafiltration is 100-120 kDa.

[0038] The present invention also provides a method for extracting the hair-growth extract, comprising the following steps:

[0039] S1, mixing saw palmetto fruit extract, ginseng root extract, caffeine, modified cationic liposome complex, and modified metal-polyphenol network nanoparticles and stirring;

[0040] S2. Make up to volume with Tris-HCl buffer and filter through a microporous filter membrane for sterilization.

[0041] The overall reaction mechanism of the hair-promoting extract described in this invention is that the comprehensive efficacy of the hair-promoting extract stems from the synergistic network of natural active ingredients, a modified delivery system, and a buffer system. This system utilizes a multi-level, multi-target regulatory strategy to precisely intervene in key stages of the hair follicle growth cycle, ultimately promoting and maintaining hair growth. Saw palmetto fruit extract and ginseng root extract, as natural plant active ingredients, provide nutritional support to hair follicles by regulating local hormone balance and improving microcirculation, respectively. The fatty acids in saw palmetto inhibit 5α-reductase activity, reducing dihydrotestosterone production and thereby slowing the miniaturization of hair follicles. Ginsenoside compounds dilate the interstitial spaces between vascular endothelial cells, increasing blood flow and oxygen supply to the hair follicle region and providing ample metabolic substrates for dermal papilla cells. Caffeine, by blocking phosphodiesterase activity and increasing intracellular cyclic adenosine monophosphate levels, prolongs the active cycle of hair follicle cells and counteracts androgen-induced shortening of the anagen phase. A modified delivery system (cationic liposome complexes and metal-polyphenol network nanoparticles) serves as an intelligent carrier for active ingredients, addressing the challenges of low bioavailability and poor targeting of natural extracts. The cationic liposome complexes utilize their positive charge to electrostatically interact with the hair follicle cell membrane, enabling efficient transmembrane transport of lipid-soluble active ingredients such as curcumin. A targeting peptide modification further directs the complex to specific receptors on the surface of hair follicle stem cells, delivering the active ingredient directly into the cell via endocytosis. This activates the Wnt / β-catenin signaling pathway and promotes the proliferation and differentiation of hair follicle stem cells. The metal-polyphenol network nanoparticles, through nanoscale size effects and surface modification, are targeted and enriched in inflamed areas of the hair follicle. The zinc oxide core provides antimicrobial protection, preventing secondary infection; the polyphenol network structure, through dual antioxidant and anti-inflammatory actions, scavenges reactive oxygen species and inhibits the release of proinflammatory cytokines, maintaining homeostasis in the hair follicle microenvironment. Hyaluronic acid oligosaccharide-mediated CD44 receptor targeting further enhances the retention of the active ingredient within the hair follicle periphery, achieving long-lasting action. The Tris-HCl buffer system serves as a stabilizing medium for the extract, providing a suitable reaction environment for each component by precisely controlling the pH and ionic strength. The weak alkaline conditions of the buffer (pH 6.4-6.6) maintain the chemical stability of the active ingredients and prevent degradation reactions caused by acidic or alkaline environments. The optimization of ionic strength ensures that the surface charge characteristics of the modified delivery system are not disturbed, maintaining its targeting function and dispersion stability. The microporous membrane filtration and sterilization step removes microbial contamination through a physical barrier to ensure the biosafety of the product. The synergistic effect of each component in the buffer system forms a dynamic equilibrium network: the natural extract provides basic nutrition and hormone regulation functions, the modified delivery system achieves precise delivery and controlled release of the active ingredients, and the buffer system maintains overall stability and functional activity.This multi-level coordinated regulatory mechanism not only promotes the proliferation and differentiation of hair follicle stem cells, but also prolongs the hair growth phase and inhibits the transition to the degeneration phase by improving the hair follicle microenvironment (anti-inflammatory, antioxidant, and antibacterial), ultimately achieving a significant increase in hair density and comprehensive improvement of hair loss problems.

[0042] According to a preferred embodiment of the present invention, in step S1, the stirring speed is 400-600 rpm, and the stirring time is 10-20 min.

[0043] According to a preferred embodiment of the present invention, in step S2, the pH of the Tris-HCl buffer is 6.4-6.6; and the pore size of the microporous filter membrane is 0.22-0.24 μm.

[0044] The beneficial effects of the present invention are:

[0045] The hair growth extract of the present invention achieves multi-target synergistic regulation of the hair follicle growth cycle through the innovative combination of two modified compounds with new structures and multiple natural active ingredients, demonstrating significant technical advantages in promoting hair growth. Modified cationic liposome complexes and modified metal-polyphenol network nanoparticles serve as core functional units, breaking through the bottlenecks of low transdermal efficiency and poor targeting of traditional hair growth preparations. The former significantly enhances the transdermal penetration of active ingredients through electrostatic interactions between the positively charged liposome structure and the negatively charged hair follicle cell membrane, enabling key active substances such as curcumin to be efficiently enriched in the hair follicle stem cell niche. The latter utilizes the stable properties of the metal-polyphenol network structure to precisely deliver natural polyphenol ingredients with antioxidant and anti-inflammatory properties to the hair follicle microenvironment, forming a long-lasting mechanism of action. The synergistic effect of this dual delivery system not only solves the problem of low bioavailability of natural ingredients, but also achieves precise enrichment of active substances in the hair follicle region through targeted positioning, laying the foundation for subsequent multi-pathway regulation.

[0046] In terms of regulating the hair follicle microenvironment, the extract of the present invention exhibits a unique multi-pathway synergistic effect. Curcumin loaded into the modified cationic liposome complex, as a key activator of the Wnt signaling pathway, can effectively stimulate the proliferation and differentiation of hair follicle stem cells and prolong the growth phase of hair; while the epigallocatechin gallate and ellagic acid in the modified metal-polyphenol network nanoparticles inhibit the activity of factors related to hair follicle miniaturization, reducing the premature transformation of hair follicles to the degeneration phase. At the same time, the saw palmetto component in the natural extract acts together on different links of the hair follicle growth cycle by regulating local hormone balance, ginseng extract improves microcirculation, and caffeine prolongs the active cycle of hair follicles. This multi-level, multi-target regulatory strategy can not only promote the generation of new hair, but also effectively prevent the loss of existing hair, improving the problem of hair loss from the root. Experimental data show that the extract has a significant effect on stimulating the proliferation of hair follicle stem cells and can significantly reduce the expression levels of markers related to hair follicle degeneration.

[0047] The technical effect of the present invention is also reflected in its excellent safety and stability. The acid-base environment of the extract is precisely adjusted by the Tris-HCl buffer system, which ensures the chemical stability of each active ingredient during storage and use, and avoids the loss of activity due to pH fluctuations. The targeting peptides and hyaluronic acid oligosaccharides modified on the surface of the modified compound not only enhance its affinity with hair follicle tissue, but also reduce the adsorption of non-specific tissues, reducing the potential risk of skin irritation. The synergistic combination of natural extracts and modified compounds has been carefully optimized, forming a complementary and synergistic mechanism while exerting their respective effects, avoiding the adverse reactions that may be caused by a single ingredient. More importantly, the extract greatly improves the utilization efficiency of active ingredients through nanoscale carrier design and molecular-level targeted modification, reduces the overall dosage requirement, and reduces the possible burden on the skin while ensuring efficacy. This formula design that takes into account both high efficiency and safety has broad prospects in clinical applications and provides an innovative solution for the treatment of hair loss. DETAILED DESCRIPTION

[0048] The following specific implementation methods are only used to further illustrate the present application and should not be understood as limiting the scope of protection of the present application. Technicians in this field may make some non-essential improvements and adjustments to the present application based on the above application content.

[0049] Example 1

[0050] A hair growth-promoting extract comprises 80g of saw palmetto fruit extract, 100g of ginseng root extract, 20g of caffeine, 20g of modified cationic liposome complex, 12g of modified metal-polyphenol network nanoparticles, and 828g of Tris-HCl buffer. Preparation of modified cationic liposome complex: 11.7g dipalmitoylphosphatidylcholine and 4.9g 1,2-dioleoyl-3-trimethylammoniopropane were weighed and dissolved in 50ml chloroform. The mixture was evaporated under reduced pressure in a water bath at 45 degrees Celsius for 60 minutes to form a uniform lipid film. 15ml phosphate buffer containing 1.2g curcumin was added to hydrate the lipid film. The liposome suspension was formed by ultrasonic treatment using a probe sonicator with a power of 200 watts for 15 minutes. The working mode of the probe sonication was set to work for 3 seconds and an interval of 2 seconds. Subsequently, 1.5g polylysine and 0.5g targeting peptide were added to the liposome suspension. The mixture was incubated at room temperature for 2 hours. The modified cationic liposome complex was obtained by separation and purification by sucrose density gradient centrifugation. The conditions for sucrose density gradient centrifugation were as follows: a sucrose gradient of 10% to 40%, a centrifugal speed of 100,000 revolutions per minute, a centrifugal temperature of 4 degrees Celsius, and a centrifugal time of 60 minutes. Preparation of modified metal-polyphenol network nanoparticles: 2.0 g of nano-zinc oxide was weighed and dispersed in 100 ml of deionized water. Ultrasonic treatment was performed for 30 minutes using a 300-watt ultrasonic instrument to form a uniform dispersion. 10.0 g of epigallocatechin gallate and 6.0 g of ellagic acid were added to the dispersion. The mixture was stirred and reacted at room temperature for 4 hours to form a metal-polyphenol network structure. Subsequently, 4.0 g of hyaluronic acid oligosaccharide was added to the reaction system and the reaction was continued for 2 hours. The retentate was collected by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 100 kilodaltons and freeze-dried. The pressure of the ultrafiltration operation was 0.1 MPa, the freeze-drying temperature was minus 50 degrees Celsius, and the freeze-drying pressure was 10 Pa. Preparation of a hair-stimulating extract: 80 g of saw palmetto fruit extract, 100 g of ginseng root extract, 20 g of caffeine, 20 g of modified cationic liposome complex, and 12 g of modified metal-polyphenol network nanoparticles were weighed and mixed. The mixture was stirred at 400 rpm for 10 minutes, and the volume was adjusted to 1000 g with Tris-HCl buffer (pH 6.4). The mixture was sterilized by filtration through a microporous filter membrane with a pore size of 0.22 μm.

[0051] Example 2

[0052] The specific implementation method is the same as that of Example 1, except that it is a hair-growth extract, comprising 100 g of saw palmetto fruit extract, 120 g of ginseng root extract, 25 g of caffeine, 25 g of modified cationic liposome complex, 15 g of modified metal-polyphenol network nanoparticles, and 815 g of Tris-HCl buffer. Preparation of modified cationic liposome complex: 14.6 g of dipalmitoylphosphatidylcholine and 6.3 g of 1,2-dioleoyl-3-trimethylammoniopropane are dissolved in chloroform, evaporated under reduced pressure to form a uniform lipid film, phosphate buffer containing curcumin is added to hydrate the lipid film, and a liposome suspension is formed by ultrasonication with a probe at a power of 205 W. Subsequently, 1.9 g of polylysine and 0.6 g of targeting peptide are added and incubated at room temperature for 3 h, and purified by sucrose density gradient centrifugation; Preparation of modified metal-polyphenol network nanoparticles: 2.5 g of nano zinc oxide is dispersed in deionized water, ultrasonicated for 35 min to form a uniform dispersion, and epigallocatechin gallate is added. 12.5 g of hyaluronan gallate and 7.5 g of ellagic acid are stirred and reacted at room temperature for 5 hours to form a metal-polyphenol network structure, followed by the addition of 5.0 g of hyaluronic acid oligosaccharide and the continued reaction for 3 hours. The retentate is collected by ultrafiltration and freeze-dried, and the molecular weight cut-off of the ultrafiltration is 110 kDa. Preparation of a hair-growth extract: Saw palmetto fruit extract, ginseng root extract, caffeine, modified cationic liposome complex, and modified metal-polyphenol network nanoparticles are mixed and stirred at 500 rpm for 15 minutes, the volume is adjusted to 1000 g with Tris-HCl buffer at a pH of 6.5, and sterilized by filtration through a microporous filter membrane with a pore size of 0.23 μm.

[0053] Example 3

[0054] The specific implementation method is the same as that of Example 1, except that, a hair-growth extract comprises 120 g of saw palmetto fruit extract, 150 g of ginseng root extract, 30 g of caffeine, 30 g of modified cationic liposome complex, 20 g of modified metal-polyphenol network nanoparticles, and 800 g of Tris-HCl buffer. Preparation of modified cationic liposome complex: 17.6 g of dipalmitoylphosphatidylcholine and 7.4 g of 1,2-dioleoyl-3-trimethylammoniopropane are dissolved in chloroform, evaporated under reduced pressure to form a uniform lipid film, phosphate buffer containing curcumin is added to hydrate the lipid film, and a liposome suspension is formed by ultrasonication with a probe having a power of 210 W, followed by addition of 2.3 g of polylysine and 0.7 g of targeting peptide, incubated at room temperature for 4 h, and purified by sucrose density gradient centrifugation; Preparation of modified metal-polyphenol network nanoparticles: 3.0 g of nano zinc oxide is dispersed in deionized water, ultrasonicated for 40 min to form a uniform dispersion, and epigallocatechin gallate is added. 15.0 g of hyaluronan gallate and 9.0 g of ellagic acid are stirred and reacted at room temperature for 6 hours to form a metal-polyphenol network structure, followed by the addition of 6.0 g of hyaluronic acid oligosaccharide and the continued reaction for 4 hours. The retentate is collected by ultrafiltration and freeze-dried, and the molecular weight cut-off of the ultrafiltration is 120 kDa. Preparation of a hair-growth extract: Saw palmetto fruit extract, ginseng root extract, caffeine, modified cationic liposome complex, and modified metal-polyphenol network nanoparticles are mixed and stirred at 600 rpm for 20 minutes, the volume is adjusted to 1000 g with Tris-HCl buffer at a pH of 6.6, and sterilized by filtration through a microporous filter membrane with a pore size of 0.24 μm.

[0055] Comparative Example 1

[0056] The specific implementation method is the same as that of Example 1, except that, an extract capable of promoting hair growth comprises 80 g of saw palmetto fruit extract, 100 g of ginseng root extract, 20 g of caffeine, 10 g of ordinary cationic liposomes, 5 g of ordinary metal-polyphenol nanoparticles, and 845 g of Tris-HCl buffer. Preparation of ordinary cationic liposomes: 11.7 g of dipalmitoylphosphatidylcholine and 4.9 g of 1,2-dioleoyl-3-trimethylammoniopropane are dissolved in chloroform, evaporated under reduced pressure to form a uniform lipid film, phosphate buffer containing curcumin is added to hydrate the lipid film, and a liposome suspension is formed by ultrasonication with a probe having a power of 200 W, which is then separated and purified by sucrose density gradient centrifugation; Preparation of ordinary metal-polyphenol nanoparticles: 2.0 g of nano zinc oxide is dispersed in deionized water, ultrasonically treated for 30 min to form a uniform dispersion, 10.0 g of epigallocatechin gallate and ellagitannin are added. 6.0 g of acid was added, stirred at room temperature for 4 hours to form a metal-polyphenol network structure, and then 4.0 g of hyaluronic acid oligosaccharide was added and the reaction was continued for 2 hours. The retentate was collected by ultrafiltration and freeze-dried, and the molecular weight cutoff of the ultrafiltration was 100 kDa. Preparation of a hair-growth extract: saw palmetto fruit extract, ginseng root extract, caffeine, ordinary cationic liposomes, and ordinary metal-polyphenol nanoparticles were mixed and stirred at 400 rpm for 10 minutes, and the volume was adjusted to 1000 g with Tris-HCl buffer at a pH of 6.4, and sterilized by filtration through a microporous filter membrane with a pore size of 0.22 μm.

[0057] Comparative Example 2

[0058] The specific implementation method is the same as that of Example 1, except that a hair-growth extract comprises 80 g of saw palmetto fruit extract, 100 g of ginseng root extract, 20 g of caffeine, 20 g of modified cationic liposome complex, and 860 g of Tris-HCl buffer. Preparation of modified cationic liposome complex: 11.7 g of dipalmitoylphosphatidylcholine and 4.9 g of 1,2-dioleoyl-3-trimethylammoniopropane were dissolved in chloroform and evaporated under reduced pressure to form a uniform lipid film. Phosphate buffer containing curcumin was added to hydrate the lipid film. The liposome suspension was formed by probe ultrasound with a power of 200 W. Subsequently, 1.5 g of polylysine and 0.5 g of targeting peptide were added and incubated at room temperature for 2 h. The suspension was purified by sucrose density gradient centrifugation. Preparation of hair-growth extract: Saw palmetto fruit extract, ginseng root extract, caffeine, and modified cationic liposome complex were mixed and stirred at 400 rpm for 10 min. The volume was adjusted to 1000 g with Tris-HCl buffer with a pH of 6.4 and sterilized by filtration through a microporous filter membrane with a pore size of 0.22 μm.

[0059] Comparative Example 3

[0060] The specific implementation method is the same as Example 1, except that a hair-growth extract includes 80g of saw palmetto fruit extract, 100g of ginseng root extract, 20g of caffeine, 12g of modified metal-polyphenol network nanoparticles, and 868g of Tris-HCl buffer. Preparation of modified metal-polyphenol network nanoparticles: 2.0 g of nano-zinc oxide was dispersed in deionized water, ultrasonically treated for 30 minutes to form a uniform dispersion, 10.0 g of epigallocatechin gallate and 6.0 g of ellagic acid were added, and the mixture was stirred and reacted at room temperature for 4 hours to form a metal-polyphenol network structure. Subsequently, 4.0 g of hyaluronic acid oligosaccharide was added and the reaction was continued for 2 hours. The retentate was collected by ultrafiltration and freeze-dried, and the molecular weight cutoff of the ultrafiltration was 100 kDa; Preparation of a hair-growth extract: Saw palmetto fruit extract, ginseng root extract, caffeine, and modified metal-polyphenol network nanoparticles were mixed and stirred at 400 rpm for 10 minutes, the volume was adjusted to 1000 g with Tris-HCl buffer with a pH of 6.4, and sterilized by filtration through a microporous filter membrane with a pore size of 0.22 μm.

[0061] Performance Testing

[0062] The hair growth extracts prepared in Examples 1-3 and Comparative Examples 1-3 were tested for performance according to the following method:

[0063] The transdermal efficiency test was conducted using an in vitro pig skin Franz diffusion cell experiment. The in vitro pig skin was fixed on the diffusion cell with the stratum corneum facing upward and the dermis in contact with the receiving solution, which was a phosphate buffer solution with a pH of 7.4. Before the experiment, the in vitro pig skin was pretreated with physiological saline for 30 minutes to remove surface impurities. 200 μL of the hair growth extract prepared in the examples and comparative examples was evenly applied to the surface of the in vitro pig skin. The effective diffusion area of ​​the diffusion cell was 0.785 cm. 2 The volume of the receiving cell was 6.5 mL, the magnetic stirrer was continuously stirred at 300 rpm, and the temperature was maintained at 37 ± 0.5 ° C. All the receiving solution was removed from the receiving cell at 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours, and the same volume was supplemented with fresh receiving solution. The caffeine content in the receiving solution was determined by high performance liquid chromatography (HPLC), and the cumulative permeation amount (μg / cm 2 ) and transdermal rate (μg / cm 2 / h), the caffeine standard curve had a good linear relationship in the range of 1-100 μg / mL, R 2≥0.999; The hair follicle proliferation activity test used an in vitro hair follicle organ culture model. Complete hair follicles were obtained from the vibrissae of C57BL / 6 mice and placed in William's E medium containing 10% fetal bovine serum. The experimental group was added with a medium containing the extracts of the examples and comparative examples (the final concentration of the extracts was equivalent to that used in vivo), while the control group was treated with only basal medium. The culture conditions were 37°C, 5% CO2 in an incubator for 7 days, with the medium replaced every 2 days. After the culture, the hair follicles were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The expression level of β-catenin was detected by immunohistochemistry, and the proportion of β-catenin nuclear translocation-positive cells, i.e., the proliferation activity index of hair follicle stem cells, was analyzed using ImageJ software; The hair follicle degeneration-related factors were detected by ELISA. Human hair follicle dermal papilla cells were cultured at 5×10 4 Cells / well were inoculated in a 6-well plate, and after culturing for 24 hours, the culture medium containing the extracts of the examples and comparative examples was replaced (the final concentration of the extracts was equivalent to that of in vivo application), and the culture was continued for 48 hours. The cell culture supernatant was collected, and the contents of IL-1α and TGF-β1 were determined according to the instructions of the ELISA kit. The operating steps were strictly in accordance with the procedures provided by the kit. The absorbance value was measured at a wavelength of 450 nm using a microplate reader, and the factor concentration was calculated using a standard curve; the animal experiment test adopted a C57BL / 6 mouse back hair removal model, and 6-8 week old male C57BL / 6 mice were selected. The hair in the back area with a diameter of 1.5 cm was removed with a depilatory cream, and the mice were randomly divided into 7 groups, 8 mice in each group, and the extracts prepared in Examples 1-3 and Comparative Examples 1-3 were applied respectively. The positive control group was applied with 2% minoxidil solution, and the negative control group was applied with an equal volume of normal saline. The application was performed once in the morning and evening every day for 21 consecutive days, and the mice were kept The living environment was the same. On the 21st day, photos of the hair loss area of ​​the mice were taken with a digital camera, and the percentage of hair coverage area was analyzed using ImageJ software. The mice were then killed, and the skin tissue of the hair loss area was taken, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The hair follicle morphology was observed by HE staining and the hair follicle length and diameter were measured. Five fields of view were randomly selected for each sample, and the average value was taken. Stability test The extracts prepared in Examples 1-3 and Comparative Examples 1-3 were stored at 4 ° C. in the dark for 30 days. Samples were taken on 0 days, 15 days, and 30 days, respectively. The contents of curcumin, epigallocatechin gallate, and ellagic acid were determined after filtration through a 0.45 μm microporous membrane. The HPLC method was used for determination. The chromatographic conditions were consistent with the active ingredient determination method in the aforementioned transdermal efficiency test. The residual rate of each active ingredient was calculated, residual rate = content after storage / initial content × 100%. At the same time, the changes in the appearance of the extracts were observed, including whether precipitation, stratification, or color change occurred.

[0064] Performance test results:

[0065] Table 1: Performance test results of various embodiments and comparative examples

[0066]

[0067]

[0068] As can be seen from Table 1, the comparison between Examples 1-3 and Comparative Examples 1-3 shows that the present invention successfully solves the technical problems of poor targeting and low efficacy of existing preparations. In terms of transdermal efficiency, the cumulative permeation of caffeine in Examples 1-3 over 24 hours was 128.7±8.3μg / cm 2 , 122.4±7.9μg / cm 2 , 118.9±6.8μg / cm 2 , while comparative examples 1-3 were 95.2±6.1μg / cm 2 , 102.8±7.4μg / cm 2 , 98.6±5.7μg / cm 2 The transdermal efficiency of the embodiment is significantly higher. This is because the modified cationic liposome complex has a positively charged liposome structure that interacts with the negatively charged hair follicle cell membrane. In addition, the polylysine increases the retention time and accurately enriches the targeted peptide, significantly improving the transdermal penetration of the active ingredient and solving the problem of poor transdermal absorption of existing preparations.

[0069] In terms of hair follicle proliferation and microenvironment regulation, the hair follicle stem cell proliferation activity (proportion of cells positive for β-catenin nuclear translocation) in Examples 1-3 was 37±2.1%, 34±1.8%, and 32±1.5%, respectively, significantly higher than the 22±1.3%, 26±1.6%, and 24±1.4% in Comparative Examples 1-3. Furthermore, the IL-1α concentrations (89.7±9.4 pg / mL to 102.4±9.1 pg / mL) and TGF-β1 concentrations (125.3±10.2 pg / mL to 138.7±12.4 pg / mL) in Examples 1-3 were significantly lower than those in Comparative Examples 1-3 (IL-1α: 142.7±11.8 pg / mL to 156.3±12.1 pg / mL; TGF-β1: 176.5±13.7 pg / mL to 189.4±14.2 pg / mL). This is due to the fact that the modified cationic liposome complex activates the Wnt / β-catenin signaling pathway to promote the proliferation of hair follicle stem cells, and the modified metal-polyphenol network nanoparticles inhibit factors related to hair follicle miniaturization, synergistically regulating the hair follicle growth cycle. Existing preparations lack this precise multi-pathway regulation capability.

[0070] Indicators such as hair coverage area, follicle length and diameter also show significant differences. The hair coverage area of ​​Examples 1-3 reached 76.5%-82.3%, the follicle length was 265-285 μm, and the follicle diameter was 63-68 μm, which was significantly better than Comparative Examples 1-3 (hair coverage area 68.5%-71.2%, follicle length 235-242 μm, follicle diameter 58-59 μm) and the negative control (hair coverage area 21.6 ± 3.2%, follicle length 180 ± 8 μm, follicle diameter 42 ± 3 μm). This shows that the extract of the present invention can more effectively promote hair growth and hair follicle development. In addition, the stability test showed that the 30-day curcumin, EGCG, and ellagic acid residue rates of Examples 1-3 were all ≥86%, indicating that they had good stability, while existing preparations may have the problem that the active ingredients are easily degraded and the efficacy is not long-lasting. In summary, the present invention solves the problems of poor targeting and low efficacy of existing preparations by synergizing natural ingredients with two modified compounds.

[0071] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A hair growth extract, characterized in that: The invention comprises the following raw materials in parts by weight: Saw palmetto fruit extract: 50-120 parts by weight; Ginseng root extract: 60-150 parts by weight; Caffeine: 10-30 parts by weight; Modified cationic liposome complex: 10-30 parts by weight; Modified metal-polyphenol network nanoparticles: 5-20 parts by weight; Tris-HCl buffer: 765-865 parts by weight; The preparation method of the modified cationic liposome complex includes: A1, dissolving dipalmitoylphosphatidylcholine and 1,2-dioleoyl-3-trimethylammoniumpropane in chloroform, evaporating under reduced pressure to form a uniform lipid film, adding a phosphate buffer containing curcumin to hydrate the lipid film, and forming a liposome suspension by probe ultrasound; A2, subsequently adding polylysine and a targeting peptide, incubating at room temperature, and separating and purifying by sucrose density gradient centrifugation.

2. The hair growth extract according to claim 1, characterized in that In step A1, the molar ratio of dipalmitoylphosphatidylcholine to 1,2-dioleoyl-3-trimethylammoniopropane is 7:3; and the power of the probe ultrasound is 200-210W.

3. The hair growth extract according to claim 1, characterized in that In step A2, the incubation time at room temperature is 2-4 hours.

4. The hair growth extract according to claim 1, characterized in that The preparation method of the modified metal-polyphenol network nanoparticles comprises: B1, dispersing nano zinc oxide in deionized water, ultrasonically treating to form a uniform dispersion, adding epigallocatechin gallate and ellagic acid, stirring and reacting at room temperature to form a metal-polyphenol network structure; B2, subsequently adding hyaluronic acid oligosaccharide to continue the reaction, collecting the retentate by ultrafiltration and freeze-drying.

5. The hair growth extract according to claim 4, characterized in that In step B1, the ultrasonic treatment time is 30-40 minutes; the molar ratio of ZnO: epigallocatechin gallate: ellagic acid is 1:5:3; and the stirring reaction time is 4-6 hours.

6. The hair growth extract according to claim 4, characterized in that In step B2, the reaction is continued for 2-4 hours; the molecular weight cut-off of ultrafiltration is 100-120 kDa.

7. A method for extracting the hair growth extract according to any one of claims 1 to 6, characterized in that the steps include: S1, mixing saw palmetto fruit extract, ginseng root extract, caffeine, modified cationic liposome complex, and modified metal-polyphenol network nanoparticles and stirring; S2. Make up to volume with Tris-HCl buffer and filter through a microporous filter membrane for sterilization.

8. The extraction method according to claim 7, characterized in that In step S1, the stirring speed is 400-600 rpm, and the stirring time is 10-20 min.

9. The extraction method according to claim 7, characterized in that In step S2, the pH of the Tris-HCl buffer is 6.4-6.6; and the pore size of the microporous filter membrane is 0.22-0.24 μm.

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