CN119280077B - Preparation and application of microspheres fully loaded with palmitoyl blue copper peptide - Google Patents

Preparation and application of microspheres fully loaded with palmitoyl blue copper peptide Download PDF

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CN119280077B
CN119280077B CN202411847006.2A CN202411847006A CN119280077B CN 119280077 B CN119280077 B CN 119280077B CN 202411847006 A CN202411847006 A CN 202411847006A CN 119280077 B CN119280077 B CN 119280077B
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polycaprolactone
palmitoyl
blue copper
copper peptide
phase solution
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CN119280077A (en
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洪民华
陈新茹
赵祥龙
任广钰
尚美彤
顾鑫欣
刘志国
唐榕
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Shanghai Paipeptide Biotechnology Co ltd
Hangzhou Peptide Biochem Co ltd
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Shanghai Paipeptide Biotechnology Co ltd
Hangzhou Peptide Biochem Co ltd
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/02Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0241Containing particulates characterized by their shape and/or structure
    • A61K8/025Explicitly spheroidal or spherical shape
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64Proteins; Peptides; Derivatives or degradation products thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/84Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
    • A61K8/85Polyesters
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/14Drugs for dermatological disorders for baldness or alopecia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/04Preparations for care of the skin for chemically tanning the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00Preparations for care of the hair
    • A61Q5/02Preparations for cleaning the hair
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00Preparations for care of the hair
    • A61Q5/12Preparations containing hair conditioners
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/56Compounds, absorbed onto or entrapped into a solid carrier, e.g. encapsulated perfumes, inclusion compounds, sustained release forms

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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  • General Chemical & Material Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention discloses preparation and application of a microsphere completely loaded with palmitoyl blue copper peptide, belongs to the field of polypeptide preparations, and particularly relates to a microsphere loaded with palmitoyl blue copper peptide. The invention adopts polyvinyl alcohol as a water-soluble polymer to prepare a water-phase solution, adopts a polycaprolactone-based polymer as an oil-soluble polymer to prepare an oil-phase solution, and prepares a sample solution from palmitoyl blue copper peptide and absolute ethyl alcohol, and then prepares the loaded palmitoyl blue copper peptide microsphere from the water-phase solution, the oil-phase solution and the sample solution together, wherein the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, the block polycaprolactone is obtained by reacting a polyhydroxy compound with absolute cyclohexanolide, the polyhydroxy compound is prepared by reacting 2, 2-dihydroxypropionic acid with ethylene glycol bishydroxymethyl ether, and the loaded palmitoyl blue copper peptide microsphere prepared by the invention has better slow release effect and the effect of improving the melanin content under the common use of the polycaprolactone and the block polycaprolactone with specific use amount.

Description

Preparation and application of microsphere completely loaded with palmitoyl blue copper peptide
Technical Field
The invention belongs to the field of polypeptide preparations, and particularly relates to preparation and application of a microsphere completely loaded with palmitoyl blue copper peptide.
Background
The amino acid sequence of the copper peptide is glycyl-L-histidyl-L-lysine, namely GHK, the small-molecule tripeptide can regulate copper ion concentration in cells, and tripeptide GHK in human blood plasma shows strong binding force with Cu 2+, and can form complex copper peptide GHK-Cu. Has effects of promoting wound healing, inhibiting inflammatory factor, promoting skin keratinocyte growth, promoting collagen synthesis, reducing photodamage, preventing alopecia, and promoting hair growth. Copper peptide is a high hydrophilic substance, most of the hydrophilic substance is insoluble in phospholipid according to the similar principle of skin compatibility, and cannot freely enter and exit cell membranes, so that the skin has limited permeation and transport capacity on the skin of the hydrophilic substance, and the bioavailability is low.
Studies have shown that GHK-Cu has poor biostability, and that when applied to wounds or damaged skin, only 0.05% to 0.15% of the GHK-Cu is absorbed, or CHK-Cu is injected into the cortex or to the wound margin, over 95% is rapidly cleared within 1 min. The half-life period of GHK-Cu is about 20min, and it is rapidly degraded and then metabolized. Thus, modification of structures to improve stability and permeability of polypeptides is a major direction of current research.
Palmitoyl blue copper peptide has the effect that the biological innovation of Zhejiang surge peptide introduces a lipophilic group palmitoyl group to carry out structural modification and reconstruction on a Lys side chain of GHK-Cu, so that a novel copper peptide Pal-GHK-Cu with a lipophilic group is successfully synthesized.
The conventional blue copper peptide has the mechanism of regulating cell proliferation and migration, reducing inflammation, promoting angiogenesis and promoting synthesis and regulation of certain proteins, is safe and low in allergy rate, and has potential application prospect in hair blackening treatment. Blue copper peptides have been shown to stimulate keratinocytes as growth factors for various differentiated cells. Studies show that GHK-Cu can stimulate in vitro hair follicle elongation, increase Bcl-2 protein expression in hair papilla cells, reduce Bax expression and promote cell proliferation. Researchers demonstrated that maintenance of GHK-Cu accelerates hair growth after human hair transplantation. Hair papilla cells are specific fibroblasts and are critical for hair follicle growth. The palmitoyl group is connected, so that short chain amino acid is protected, and the lipophilicity and stability of GHK-Cu are improved. Proved by researches, the palmitoyl blue copper peptide has better bioavailability, stability and application safety compared with the copper peptide. The dual characteristics of Pal-GHK show that the peptide has wide application prospect in skin cell research and great potential in strengthening extracellular matrix. In addition, the palmitoyl modified blue copper peptide not only has the conventional hair-growing capability, but also has the outstanding effects of blacking, white hair and the like by activating a tyrosinase signal channel. The scalp nourishing and blackening hair-generating composition has a composite effect for middle-aged and elderly people. No component has the effects at the same time. However, the ideal active ingredient needs to have not only specificity and pharmacological activity but also sufficient reach the target site to act. The effect of palmitoyl blue copper peptide alone is difficult to achieve.
The microsphere is a skeleton type microspherical entity formed by dissolving or dispersing an active substance in a high polymer material, and the particle size range of the microsphere is 1-250 mu m. The biodegradable polymer is used as a conveying carrier of the polypeptide, the polypeptide is embedded, the stability of the medicine can be improved, the toxic and side effects of the medicine on surrounding tissues and non-target tissue cells can be reduced in a targeted manner, and the like. In addition, the targeting of the microspheres can concentrate the active ingredients in a target area, control the release speed, prolong the action time and improve the curative effect. The microsphere has unique advantages as a novel carrier, has great development potential, and is one of hot spots for research on sustained and controlled release preparations in recent years. The palmitoyl blue copper peptide can be loaded in the microsphere, and can be endowed with more application scenes of medical appliances and cosmetics.
Numerous microsphere preparation patents are currently available, mostly for the loading of hydrophilic active ingredients, like water-soluble polypeptides, requiring a W/O/W system, complicated preparation process and limited encapsulation efficiency, CN111836654a discloses a polycaprolactone microsphere filler comprising vitamin C and a preparation method thereof, specifically comprising (a) preparing a dispersed phase by dissolving polycaprolactone in a first solvent and vitamin C in a second solvent, and then uniformly mixing the two solutions to prepare a single solution, (b) preparing an emulsion by mixing the dispersed phase with an aqueous solution (continuous phase) comprising a surfactant, (C) forming microspheres by extracting an organic solvent from the dispersed phase in the emulsion prepared in step (b) into the continuous phase and evaporating, and (d) recovering the microspheres from the continuous phase of step (C) to prepare polycaprolactone microspheres. Compared with a single emulsion method, the method is more complicated, particularly in the production process, and the encapsulation efficiency is lower than that of the single emulsion method. The burst release phenomenon can occur in the later period of the slow release process, which is unfavorable for the action of the encapsulating ingredient. CN118121719a discloses a medicine slow-release porous silica gel material, a preparation method and application thereof, the method is that porous microspheres are prepared, firstly, the microspheres are prepared by using a placeholder, then the placeholder is melted off, and finally, the microspheres are immersed in an aqueous solution to adsorb target polypeptide. The method has low loading rate, serious waste of valuable medicaments, more adsorption among the porous medicaments is under the action of Van der Waals force, and the release of the valuable medicaments is easy in the process of storing the medicaments, so that the storage challenge is very large. The initial entry of the porous loaded microspheres into the human body is likely to cause the drug burst phenomenon.
Disclosure of Invention
The invention aims to provide a preparation and application of a microsphere completely loaded with palmitoyl blue copper peptide, which has good stability, good loading effect, good encapsulation effect and good slow release effect.
The technical scheme adopted by the invention for achieving the purpose is as follows:
A preparation method of palmitoyl blue copper peptide-loaded microspheres comprises the following steps:
mixing a sample solution with an oil phase solution, adding a water phase solution, mixing to form emulsion, performing vacuum sealing treatment, and finally performing separation treatment to obtain palmitoyl blue copper peptide-loaded microspheres;
the sample solution contains palmitoyl blue copper peptide, the oil phase solution contains oil-soluble polymer, the oil-soluble polymer comprises at least 1 of polycaprolactone-based polymer, polylactic acid-based polymer, polyhydroxyalkanoate-based polymer and polyglycolide-lactide copolymer, and the polycaprolactone-based polymer comprises polycaprolactone and/or block polycaprolactone;
The block polycaprolactone has a polyhydroxy structure formed by 2, 2-dihydroxypropionic acid and ethylene glycol bishydroxymethyl ether, and a polycaprolactone structure. The block polycaprolactone has a branched chain structure, the polycaprolactone is of a linear structure, and in an optional range, the block polycaprolactone and the polycaprolactone are used as oil-soluble polymers, so that a coating site for palmitoyl blue copper peptide can be provided in the process of mutually crossing and winding, palmitoyl blue copper peptide can be better loaded, the stability of palmitoyl blue copper can be improved, and the toxic and side effects of palmitoyl blue copper on surrounding tissues and non-target tissue cells can be reduced in a targeted manner. The targeting property of the palmitoyl blue copper peptide-loaded microsphere can also enable the active ingredient to be concentrated in a target area, delay the release speed of palmitoyl blue copper peptide, prolong the action time and improve the curative effect, even if the release speed is slower, the palmitoyl blue copper peptide-loaded microsphere is better concentrated in the target area under the coating of the block polycaprolactone and the polycaprolactone, and the effect of the palmitoyl blue copper peptide-loaded microsphere is improved.
Preferably, the solvent of the sample solution is absolute ethanol, or the solvent of the oil phase solution is DCM, or the water phase solution contains water-soluble polymer and water.
More preferably, the content of palmitoyl blue copper peptide in the sample solution is 2-5000ppm, or the content of the oil-soluble polymer in the oil phase solution is 1-25wt%, or the water-soluble polymer comprises polyvinylpyrrolidone or polyvinyl alcohol, or the content of the water-soluble polymer in the aqueous phase solution is 0.1-1.2wt%.
Preferably, the volume ratio of the sample solution to the oil phase solution is 1:100-1:5, or the volume ratio of the sample solution to the aqueous phase solution is 1:1000-1:25.
Preferably, the oil-soluble polymer is a polycaprolactone-based polymer, including polycaprolactone and blocked polycaprolactone.
More preferably, the polycaprolactone and blocked polycaprolactone are used in an amount of 1:0.5 to 3 in the polycaprolactone-based polymer.
Preferably, in the process of mixing to form emulsion, homogenizing treatment is adopted to form emulsion, and vacuum sealing treatment is adopted to vacuumize for 2-6h by adopting a water pump.
Preferably, in the separation treatment, the product of the vacuum sealing treatment is subjected to filtration, centrifugation and freeze-drying treatment.
Preferably, the preparation of the palmitoyl blue copper peptide-loaded microsphere can also comprise the preparation of polyhydroxy compounds and the preparation of block polycaprolactone.
More preferably, in the preparation of the polyhydroxy compound, 2-dihydroxypropionic acid and ethylene glycol bis-hydroxymethyl ether are mixed, a catalyst, a water-carrying agent and a polymerization inhibitor are added for reaction for 6-24 hours at the temperature of 100-130 ℃, the reaction is adjusted to be neutral after the reaction is finished, and the polyhydroxy compound is obtained by washing with saturated sodium chloride solution and distilling under reduced pressure.
More preferably, in the preparation of the polyhydroxy compound, ethylene glycol bis-hydroxymethyl ether is used in an amount of 40 to 60% by weight of 2, 2-dihydroxypropionic acid.
More preferably, in the preparation of the polyhydroxy compound, the catalyst is cerium sulfate, and the catalyst is used in an amount of 0.5 to 3wt% of 2, 2-dihydroxypropionic acid.
More preferably, in the preparation of the polyhydroxy compound, the water-carrying agent is cyclohexane, and the use amount of the water-carrying agent is 80-120wt% of 2, 2-dihydroxypropionic acid.
More preferably, in the preparation of the polyhydroxy compound, the polymerization inhibitor is hydroquinone and the polymerization inhibitor is used in an amount of 1 to 3wt% of 2, 2-dihydroxypropionic acid.
More preferably, in the preparation of the block polycaprolactone, the polyhydroxy compound and the anhydrous polycaprolactone are mixed, the catalyst is added, the reaction is carried out for 10-48 hours at 100-130 ℃ under the nitrogen atmosphere, after the reaction is completed, the cooling is carried out, the dichloromethane is added, the glacial ethyl ether is then added, the precipitation is carried out, and the block polycaprolactone is obtained after filtration and drying.
More preferably, in the preparation of the blocked polycaprolactone, the polyhydroxy compound is used in an amount of 10 to 30% by weight of the anhydrous polycaprolactone.
More preferably, in the preparation of the block polycaprolactone, the catalyst is stannous octoate, and the catalyst is used in an amount of 0.2-2wt% of the anhydrous polycaprolactone.
More preferably, in the preparation of the block polycaprolactone, dichloromethane is used for dissolving the crude product, and glacial diethyl ether is used for precipitating and precipitating, and the dichloromethane and the glacial diethyl ether are used in proper amounts.
Preferably, in the preparation of the aqueous phase solution, the water-soluble polymer is added into water, and dissolved and cooled at 80-90 ℃ to obtain the aqueous phase solution.
More preferably, the water-soluble polymer comprises polyvinylpyrrolidone or polyvinyl alcohol in the preparation of the aqueous solution, and the content of the water-soluble polymer in the aqueous solution is 0.1 to 1.2wt%.
Preferably, in the preparation of the oil phase solution, the oil-soluble polymer is added into the oil phase solvent, and is dissolved by shaking to obtain the oil phase solution.
More preferably, in the preparation of the oil phase solution, the oil-soluble polymer includes at least 1 of a polycaprolactone-based polymer, a polylactic acid-based polymer, a polyhydroxyalkanoate-based polymer, and a polyglycolide-lactide copolymer.
More preferably, in the preparation of the oil phase solution, the oil phase solvent includes at least 1 of DCM, DMF and DMSO.
More preferably, the oil phase solution is prepared such that the oil soluble polymer is present in the oil phase solution in an amount of 1 to 25wt%.
More preferably, in the preparation of the oil phase solution, the polycaprolactone-based polymer comprises polycaprolactone and/or blocked polycaprolactone.
More preferably, in the preparation of the oil phase solution, the polycaprolactone and the blocked polycaprolactone are used in an amount of 1:0.5-3 in the polycaprolactone-based polymer.
Preferably, in the preparation of the sample solution, the sample is added into the sample solvent, and is dissolved by shaking to obtain the sample solution.
More preferably, in the preparation of the sample solution, the sample comprises palmitoyl blue copper peptide, the sample solvent comprises absolute ethyl alcohol, and the content of the sample in the sample solution is 2-5000ppm.
Preferably, in the preparation of the palmitoyl blue copper peptide-loaded microsphere, a sample solution and an oil phase solution are mixed, then an aqueous phase solution is added, and the mixture is homogenized to form a blue copper peptide emulsion, and the blue copper peptide-loaded microsphere is obtained through vacuum treatment for 2-6 hours after sealing, filtration, washing and freeze-drying.
More preferably, in the preparation of the palmitoyl blue copper peptide-loaded microsphere, the volume ratio of the sample solution to the oil phase solution is 1:100-1:5.
More preferably, in the preparation of the palmitoyl blue copper peptide-loaded microsphere, the volume ratio of the sample solution to the aqueous phase solution is 1:1000-1:25.
Preferably, sucrose polyoxypropylene ether can be added into the aqueous phase solution for preparing the invention, and the use amount of the sucrose polyoxypropylene ether is 0.5-4wt% of the water-soluble polymer. The invention can further use polyvinyl alcohol and sucrose polyoxypropylene ether together in aqueous phase solution, the polyvinyl alcohol and the sucrose polyoxypropylene ether can lead the palmitoyl blue copper peptide microsphere to be more reasonably coated in microsphere sites formed by polycaprolactone and block polycaprolactone, thereby slowing the release speed of palmitoyl blue copper peptide in the obtained palmitoyl blue copper peptide-carrying microsphere, but ensuring that palmitoyl blue copper peptide is better concentrated in a target area and improving the effect of palmitoyl blue copper peptide-carrying microsphere.
The invention discloses palmitoyl blue copper peptide-loaded microspheres prepared by the preparation method.
The invention discloses application of the palmitoyl blue copper peptide-loaded microsphere in preparing shampoo products and/or hair care products and/or scalp injection nourishing products and/or hair growth and hair loss prevention products and/or blackness products.
The invention adopts the polyvinyl alcohol as a water-soluble polymer to prepare a water-phase solution, adopts the polycaprolactone-based polymer as an oil-soluble polymer to prepare an oil-phase solution, and prepares the palmitoyl blue copper peptide-loaded microsphere by the water-phase solution, the oil-phase solution and the sample solution together, wherein the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, the block polycaprolactone is obtained by reacting a polyhydroxy compound and anhydrous cyclohexane, and the polyhydroxy compound is prepared by reacting 2, 2-dihydroxypropionic acid and ethylene glycol bishydroxymethyl ether, so that the palmitoyl blue copper peptide-loaded microsphere has better slow release effect and the effect of improving the melanin content under the common use of the polycaprolactone with specific use amount and the block polycaprolactone. Therefore, the invention is the preparation and application of the microsphere with the complete load of palmitoyl blue copper peptide, which has good stability, good loading effect, good encapsulation effect and good slow release effect.
Drawings
FIG. 1 is an electron microscope image of palmitoyl blue copper peptide-loaded microspheres.
FIG. 2 is a graph showing relative gene expression in hair papilla cells.
FIG. 3 is a graph showing relative gene expression of A375 cells.
FIG. 4 is a graph showing melanin content.
Fig. 5 is a graph of release rate.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The concept of the present application will be described with reference to the accompanying drawings. It should be noted that the following descriptions of the various concepts are only for making the content of the present application easier to understand, and are not meant to limit the scope of the present application, and at the same time, the embodiments of the present application and the features in the embodiments may be combined with each other without conflict. The application will be described in detail below with reference to the drawings in connection with embodiments.
Example 1 preparation method of palmitoyl blue copper peptide-loaded microspheres
And (3) preparing an aqueous phase solution, namely adding the water-soluble polymer into water, and dissolving and cooling at 85 ℃ to obtain the aqueous phase solution. The water-soluble polymer included polyvinyl alcohol, and the content of the water-soluble polymer in the aqueous phase solution was 0.6wt%.
The preparation of the oil phase solution comprises the steps of adding an oil-soluble polymer into an oil phase solvent, and oscillating and dissolving to obtain the oil phase solution. The oil-soluble polymer is a polycaprolactone-based polymer, and the polycaprolactone-based polymer is polycaprolactone. The oil phase solvent was DCM. The content of the oil-soluble polymer in the oil phase solution was 15wt%.
And (3) preparing a sample solution, namely adding the sample into a sample solvent, and oscillating for dissolving to obtain the sample solution. The sample is palmitoyl blue copper peptide, the sample solvent is absolute ethyl alcohol, and the content of the sample in the sample solution is 1000ppm.
And (3) preparing the palmitoyl blue copper peptide-loaded microspheres, namely mixing a sample solution with an oil phase solution, adding the water phase solution, homogenizing to form blue copper peptide emulsion, sealing, performing vacuum treatment for 4 hours, filtering, washing and freeze-drying to obtain the palmitoyl blue copper peptide-loaded microspheres. The volume ratio of the sample solution to the oil phase solution is 1:10, and the volume ratio of the sample solution to the water phase solution is 1:100.
Example 2 preparation method of palmitoyl blue copper peptide-loaded microspheres
The difference between this example and example 1 is that the oil-phase solution is prepared in which the oil-soluble polymer is a polycaprolactone-based polymer, the polycaprolactone-based polymer is a polycaprolactone and a block polycaprolactone, and the amount of polycaprolactone and block polycaprolactone used in the polycaprolactone-based polymer is 1:0.8.
The preparation of polyhydroxy compound includes mixing 2, 2-dihydroxypropionic acid and glycol bis-hydroxymethyl ether, adding catalyst, water carrying agent and polymerization inhibitor, reacting at 120 deg.c for 12 hr, regulating to neutrality, washing with saturated sodium chloride solution, and vacuum distilling to obtain polyhydroxy compound. The consumption of ethylene glycol bis-hydroxymethyl ether is 50wt% of 2, 2-dihydroxypropionic acid, the catalyst is cerium sulfate, the consumption of the catalyst is 1.5wt% of 2, 2-dihydroxypropionic acid, the water-carrying agent is cyclohexane, the consumption of the water-carrying agent is 100wt% of 2, 2-dihydroxypropionic acid, the polymerization inhibitor is hydroquinone, and the consumption of the polymerization inhibitor is 2wt% of 2, 2-dihydroxypropionic acid.
The preparation of the block polycaprolactone comprises the steps of mixing polyhydroxy compound and anhydrous cyclohexane, adding a catalyst, reacting for 24 hours at 120 ℃ under the nitrogen atmosphere, cooling after the reaction is finished, adding dichloromethane, adding glacial ethyl ether, precipitating, filtering, and drying to obtain the block polycaprolactone. The usage amount of the polyhydroxy compound is 20wt% of the anhydrous cyclohexane, the catalyst is stannous octoate, and the usage amount of the catalyst is 1.2wt% of the anhydrous cyclohexane. The dichloromethane is used to dissolve the crude product, the glacial ethyl ether is used to precipitate, and a proper amount of dichloromethane and glacial ethyl ether can be used.
Example 3 preparation method of palmitoyl blue copper peptide-loaded microspheres
This example differs from example 2 in that the oil phase solution is prepared in which the oil-soluble polymer is a polycaprolactone-based polymer and the polycaprolactone-based polymer is a polycaprolactone and the polycaprolactone-block, and the amount of polycaprolactone and polycaprolactone-block used is 1:2.4.
Example 4 preparation method of palmitoyl blue copper peptide-loaded microspheres
This example is different from example 3 in that a water phase solution is prepared, and sucrose polyoxypropylene ether is further added to the water phase solution, wherein the sucrose polyoxypropylene ether is used in an amount of 0.8wt% of the water-soluble polymer.
Example 5 preparation method of palmitoyl blue copper peptide-loaded microsphere
This example differs from example 4 in the preparation of an aqueous phase solution to which sucrose polyoxypropylene ether was also added, the sucrose polyoxypropylene ether being used in an amount of 3.2% by weight of the water-soluble polymer.
Comparative example 1 preparation method of palmitoyl blue copper peptide-loaded microsphere
This example differs from example 1 in that the oil phase solution was prepared in which the oil-soluble polymer was a polycaprolactone-based polymer, which was a block polycaprolactone, and the block polycaprolactone was synthesized as in example 2.
Comparative example 2 preparation method of palmitoyl blue copper peptide-loaded microsphere
The difference between this example and example 2 is that the oil-soluble polymer is a polycaprolactone-based polymer, the polycaprolactone-based polymer is a polycaprolactone and a block polycaprolactone, and the amount of polycaprolactone and block polycaprolactone used in the polycaprolactone-based polymer is 1:0.3.
The specific proportion has good effect
Comparative example 3 preparation method of palmitoyl blue copper peptide-loaded microsphere
This example differs from example 2 in that the oil phase solution is prepared in which the oil-soluble polymer is a polycaprolactone-based polymer and the polycaprolactone-based polymer is a polycaprolactone and the polycaprolactone-block, and the amount of polycaprolactone and polycaprolactone-block used is 1:3.5.
Test example:
The invention carries out electron microscope characterization on the palmitoyl blue copper peptide-loaded microsphere prepared in the embodiment 1, and the result is shown in figure 1, and the particle size of the palmitoyl blue copper peptide-loaded microsphere prepared by the method in the embodiment 1 is 10-50 mu m.
1. Regulation and control test of anti-drop hair-fixing gene
The influence of palmitoyl blue copper peptide microspheres on the expression of the hair papilla cell anti-hair loss and hair fixation related genes is analyzed by utilizing a Luminex200 suspension chip multiple detection system technology and combining QGP methods.
The experiment used papilla cells plated in 24 well plates at a density of 6 x10 4 cells per well, 0.5 mL per well volume. Cell well plates were cultured in an incubator 24 h. The experiment is divided into a negative control and a sample group, wherein each group comprises 3 compound holes, and the sample group is the palmitoyl blue copper peptide-loaded microsphere prepared in the example 1.
The invention detects the effect of palmitoyl blue copper peptide-loaded microspheres prepared in the embodiment 1 on the expression of 25 genes in hair papilla cells, wherein the 2 genes are FGF7、VEGFA、LPAR6、EDAR、IGF1、ITGA6、KRT75、FOXL2、VDR、SOX21、WNT4、BMP3、COL17A1、TGM3、TGFB1、DKK1、IL6、AR、TRPV3、SRD5A2、TNF、ESR1、ACE2、TBP、HPRT1, respectively, have promotion effect on the expression of FGF7, IGF1, KRT75, VDR, COL17A1 and other genes, and can down regulate the relative expression quantity of DKK1 and IL6 genes. As shown in FIG. 2, NC is a negative control group, and the sample is palmitoyl blue copper peptide-loaded microsphere prepared in example 1, compared with the negative control group, the sample group can significantly up-regulate relative expression amounts of FGF7, IGF1, FOXL2, WNT4 and BMP3 genes (p < 0.01), and can significantly down-regulate relative expression amounts of DKK1 and IL6 genes (p < 0.01). The result shows that the palmitoyl blue copper peptide-loaded microsphere prepared by the invention has an adjusting effect on partial anti-hair loss and hair fixing genes.
According to current studies, FGF7 is a member of the Fibroblast Growth Factor (FGF) family. FGF family members are involved in a variety of biological processes such as embryonic development, cell growth, morphogenesis, and tissue repair. FGF7 has an important role in wound re-epithelialization, hair development, and early lung organogenesis. Dickkopf related protein (DKKI) is an important antagonist of the Wnt signaling pathway, which plays an important regulatory role in hair follicle development and regeneration. When DKK1 is continuously over-expressed during embryo development, hair follicles cannot be generated, and when DKK1 is over-expressed after birth, the growth of the hair follicles is inhibited. In addition, interleukins (ILs) include IL-1a, IL-1B, IL-6, etc., which exert effects on the extracellular matrix of dermal papilla and intracellular cAMP levels that regulate hair follicle growth. ILs stimulation causes hair follicle malnutrition in anagen phase such as deformation and shrinkage of hair papilla, abnormal cavitation of hair matrix cells and abnormal keratosis of hair bulb, and can inhibit hair follicle and hair growth.
2. Test of blackening promoting Gene
The influence of palmitoyl blue copper peptide-loaded microspheres prepared in example 1 on the gene expression in melanoma cells A375 is analyzed by adopting QGP method of Luminex200 suspension chip multiplex detection system.
The present invention uses a375 cells plated in 24 well plates at a density of 6 x 10 4 cells per well, 0.5 mL per well volume. Cell well plates were cultured in an incubator 24 h. The experiment is divided into a negative control and a sample group, wherein each group comprises 3 compound holes, and the sample group is the palmitoyl blue copper peptide-loaded microsphere prepared in the example 1.
The study totally detects the expression of 36 genes, wherein 36 genes are PTGS2、PTGER1、CREB1、ASIP、EDN3、COMMD3、MET、MCIR、HGF、DCT、F2RL1、OCA2、IL1RN、KIT、NQO1、HPRT1、TP53、CNR1、LEF1、MITF、RAB27A、PMEL、TNF、SLC45A2、IL1A、KLF6、TBP、CPX3、DSG1、LICAM、TYR、POMC、SOX10、TYRP1、TGFB1、CDSN, respectively, and the palmitoyl blue copper peptide-loaded microsphere prepared in the embodiment 1 has a regulating effect on related genes such as TYP, KIT, POMC, PMEL, OCA2, PTGS2, IL1A and the like in melanoma cells A375. As shown in FIG. 3, NC is a negative control group, and the sample is the palmitoyl blue copper peptide-loaded microsphere prepared in example 1, and compared with the negative control group, the palmitoyl blue copper peptide-loaded microsphere prepared in example 1 can significantly up-regulate the relative expression amount of DCT and POMC genes (P < 0.01), the relative expression amount of EDN3 and TYR genes (P < 0.05), and can significantly reduce the relative expression amount of IL1A genes (P < 0.01). The result shows that the palmitoyl blue copper peptide-loaded microsphere prepared by the invention can regulate the expression quantity of different related genes in melanoma cells A375.
In conclusion, the palmitoyl blue copper peptide-loaded microsphere prepared by the invention can change the intracellular gene expression level when being used for treating melanoma cells A375, and has promotion and inhibition effects on melanin synthesis related genes such as DCT, tyrosinase activity related genes such as TYP and other related genes.
3. Melanin content test
The invention explores the influence of palmitoyl blue copper peptide-loaded microspheres on the content of melanin in cells through human melanoma cells A375.
The experiment is divided into a negative control group, a positive control group and a sample group, wherein the negative control group is a complete DMEM medium without any treatment, the positive control group is a complete DMEM medium containing 1mg/mL kojic acid, and the sample group is a complete DMEM medium containing palmitoyl blue copper peptide microspheres prepared in examples 1-5 or comparative examples 1-3.
After the palmitoyl blue copper peptide-loaded microspheres are applied to human melanoma cells A375, the content of melanin in the human melanoma cells A375 is shown as a figure 4, wherein NC is a negative control group, PC is a positive control group, S1 is example 1, S2 is example 2, S3 is example 3, S4 is example 4, S5 is example 5, D1 is comparative example 1, D2 is comparative example 2, D3 is comparative example 3, and compared with the NC group, the content of melanin in the human melanoma cells A375 treated by the palmitoyl blue copper peptide-loaded microspheres of example 1 is obviously increased, so that the content of melanin in the human melanoma cells A375 is obviously increased, and the content of melanin in the human melanoma cells A375 in the PC group is greatly reduced, compared with example 1, the prepared palmitoyl blue peptide-loaded microspheres have the effect of improving the content of polycaprolactone and the polycaprolactone in the polycaprolactone group, and the polycaprolactone in the polycaprolactone group is better than that the polycaprolactone group is used in the polycaprolactone group, and the polycaprolactone group is 3 is used, and the polycaprolactone-containing the polycaprolactone-3 is used singly, and the effect of the polycaprolactone-based on the polycaprolactone-3 is better than the effect of the polycaprolactone-based on the polycaprolactone-A-3. Examples 4 to 5 compared with example 3 show that when preparing the palmitoyl blue copper peptide-loaded microspheres, sucrose polyoxypropylene ether can be added when polyvinyl alcohol exists in the aqueous phase solution, and after the palmitoyl blue copper peptide-loaded microspheres are prepared by the oil phase solution and the sample solution together with the polyvinyl alcohol and the sucrose polyoxypropylene ether, the content of melanin in human melanoma cells A375 can be further improved, and the performance of the palmitoyl blue copper peptide-loaded microspheres can be improved by the joint use of the polyvinyl alcohol and the sucrose polyoxypropylene ether.
4. Release Effect test of palmitoyl blue copper peptide
The test sample in the experiment is the palmitoyl blue copper peptide-loaded microsphere prepared in the examples 1-5 or the comparative examples 1-4, 0.2 g of the test sample is accurately weighed, placed in a 15mL centrifuge tube containing 9.8gPSB of solution, sealed and placed in a shaking incubator for constant-temperature shaking at 37 ℃ and 150 r/min. And (3) centrifugally sampling for 5mL hours, and detecting the polypeptide content in the sample by utilizing high performance liquid chromatography. The release rate of the palmitoyl blue copper peptide-loaded microspheres was calculated.
The test results of the release of the palmitoyl blue copper peptide are shown in fig. 5, wherein S1 is example 1, S2 is example 2, S3 is example 3, S4 is example 4, S5 is example 5, D1 is comparative example 1, D2 is comparative example 2, D3 is comparative example 3, and examples 2-3 are compared with example 1, and show that the prepared palmitoyl blue copper peptide-loaded microsphere has higher slow release effect when polycaprolactone and block polycaprolactone are jointly adopted as oil-soluble polymers, and the increase of the ratio of the block polycaprolactone and the slow release effect of the palmitoyl blue copper peptide-loaded microsphere can be improved when the polycaprolactone and the block polycaprolactone are used, and examples 1-3 are compared with comparative examples 1-3, and are superior to the use of polycaprolactone or the block polycaprolactone alone, and the use amount of polycaprolactone and the block polycaprolactone satisfy the specific proportion relationship and only have the effect of improving the slow release effect of the palmitoyl blue peptide-loaded microsphere. Examples 4 to 5 compared with example 3 show that when the palmitoyl blue copper peptide-loaded microspheres are prepared, sucrose polyoxypropylene ether can be added when polyvinyl alcohol exists in the aqueous phase solution, and after the palmitoyl blue copper peptide-loaded microspheres are prepared by the combination of the polyvinyl alcohol and the sucrose polyoxypropylene ether and the oil phase solution and the sample solution, the slow release effect of the palmitoyl blue copper peptide-loaded microspheres can be further improved, and the use of the polyvinyl alcohol and the sucrose polyoxypropylene ether can also improve the slow release performance of the palmitoyl blue copper peptide-loaded microspheres.
The above examples and/or embodiments are merely for illustrating the preferred embodiments and/or implementations of the present technology, and are not intended to limit the embodiments and implementations of the present technology in any way, and any person skilled in the art should be able to make some changes or modifications to the embodiments and/or implementations without departing from the scope of the technical means disclosed in the present disclosure, and it should be considered that the embodiments and implementations are substantially the same as the present technology.
The principles and embodiments of the present application have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present application and its core ideas. The foregoing is merely illustrative of the preferred embodiments of the application, and it will be appreciated that numerous modifications, adaptations and variations of the application can be made by those skilled in the art without departing from the principles of the application, and that other features and advantages of the application can be combined in any suitable manner, and that no improvement in the design or design of the application is intended to be applied directly to other applications.

Claims (8)

1.一种负载棕榈酰蓝铜肽微球的制备方法,包括:1. A method for preparing palmitoyl blue copper peptide loaded microspheres, comprising: 将样品溶液与油相溶液混合,然后加入水相溶液混合形成乳液,真空密封处理,最后经分离处理得到负载棕榈酰蓝铜肽微球;所述样品溶液中棕榈酰蓝铜肽的含量为2-5000ppm;The sample solution is mixed with the oil phase solution, and then the aqueous phase solution is added to form an emulsion, vacuum-sealed, and finally separated to obtain microspheres loaded with palmitoyl blue copper peptide; the content of palmitoyl blue copper peptide in the sample solution is 2-5000 ppm; 所述样品溶液中含有棕榈酰蓝铜肽;所述油相溶液中含有油溶性聚合物,所述油溶性聚合物为聚己内酯基聚合物,聚己内酯基聚合物包括聚己内酯和嵌段聚己内酯;所述聚己内酯基聚合物中聚己内酯和嵌段聚己内酯的使用量为1:0.5-3;The sample solution contains palmitoyl blue copper peptide; the oil phase solution contains an oil-soluble polymer, the oil-soluble polymer is a polycaprolactone-based polymer, and the polycaprolactone-based polymer includes polycaprolactone and block polycaprolactone; the usage amount of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:0.5-3; 所述嵌段聚己内酯中具有2,2-二羟基丙酸和乙二醇双羟甲基醚形成多羟基结构,以及聚己内酯的结构;The block polycaprolactone has a polyhydroxy structure formed by 2,2-dihydroxypropionic acid and ethylene glycol bis(hydroxymethyl)ether, and a polycaprolactone structure; 所述水相溶液中含有水溶性聚合物和水,所述水溶性聚合物包括聚乙烯吡咯烷酮或聚乙烯醇。The aqueous phase solution contains a water-soluble polymer and water, and the water-soluble polymer includes polyvinyl pyrrolidone or polyvinyl alcohol. 2.根据权利要求1所述的一种负载棕榈酰蓝铜肽微球的制备方法,其特征在于:所述样品溶液的溶剂为无水乙醇;或,所述油相溶液中的溶剂为DCM。2. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 1, characterized in that: the solvent of the sample solution is anhydrous ethanol; or the solvent in the oil phase solution is DCM. 3.根据权利要求2所述的一种负载棕榈酰蓝铜肽微球的制备方法,其特征在于:所述油相溶液中油溶性聚合物的含量为1-25wt%;或,水相溶液中水溶性聚合物的含量为0.1-1.2wt%。3. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 2, characterized in that: the content of the oil-soluble polymer in the oil phase solution is 1-25wt%; or, the content of the water-soluble polymer in the water phase solution is 0.1-1.2wt%. 4.根据权利要求1所述的一种负载棕榈酰蓝铜肽微球的制备方法,其特征在于:所述样品溶液与油相溶液的体积比为1:100-1:5;或,样品溶液与水相溶液的体积比为1:1000-1:25。4. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 1, characterized in that the volume ratio of the sample solution to the oil phase solution is 1:100-1:5; or the volume ratio of the sample solution to the aqueous phase solution is 1:1000-1:25. 5.根据权利要求1所述的一种负载棕榈酰蓝铜肽微球的制备方法,其特征在于:所述混合形成乳液中,采用均质处理形成乳液;或,所述真空密封处理采用水泵抽真空;或,所述真空密封处理的时间为2-6h。5. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 1, characterized in that: in the mixing to form the emulsion, a homogenization treatment is used to form the emulsion; or, the vacuum sealing treatment is performed by vacuuming with a water pump; or, the vacuum sealing treatment lasts for 2-6 hours. 6.根据权利要求1所述的一种负载棕榈酰蓝铜肽微球的制备方法,其特征在于:所述分离处理中,将真空密封处理的产物经过滤,离心,冻干处理。6. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 1, characterized in that: in the separation process, the vacuum sealed product is filtered, centrifuged, and freeze-dried. 7.权利要求1-6任一所述制备方法制备得到的负载棕榈酰蓝铜肽微球。7. Palmitoyl blue copper peptide loaded microspheres prepared by any one of the preparation methods of claims 1-6. 8.权利要求7所述的负载棕榈酰蓝铜肽微球在制备洗发用品和/或护发用品和/或头皮注射滋养产品和/或生发防脱产品和/或美黑用品中的用途。8. Use of the palmitoyl blue copper peptide loaded microspheres according to claim 7 in the preparation of shampoo products and/or hair care products and/or scalp injection nourishing products and/or hair growth and anti-hair loss products and/or tanning products.
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