KR20230153562A - A complex with epidermal growth factor and use thereof - Google Patents

Abstract

Relates to epithelial cell growth factor complex and its uses. The reinforced epithelial cell growth factor fusion complex combined with zinc according to the present invention promotes dimerization of the epithelial cell growth factor receptor and effectively supplies zinc into cells, significantly improving the proliferation ability and wound healing ability of keratinocytes. I confirmed what I ordered. Therefore, it is expected that the composition containing the reinforcing epithelial cell growth factor can be used for skin regeneration, prevention, improvement, or treatment of damage.

Description

Epidermal growth factor complex and use thereof {A complex with epidermal growth factor and use thereof}

Relates to epithelial cell growth factor complex and its uses.

Skin is a body tissue that exists in the outermost layer of the human body. It is an important element that protects the body from the external environment through various physiological functions and is also responsible for externally visible aesthetic functions. As the skin ages, not only does skin immune function deteriorate, but also external deformities such as decreased skin elasticity, changes in skin color, and formation of skin wrinkles occur. This skin aging process can be classified into intrinsic aging and extrinsic aging. Intrinsic aging is natural aging that occurs naturally as we age, and is mainly influenced by genetic factors, so it is difficult to control artificially. On the other hand, extrinsic aging occurs due to an increase in free radicals in the body due to environmental factors. Relatively artificial control is possible.

Human epidermal growth factor (EGF) is a protein that promotes cell proliferation and differentiation by binding to the epidermal growth factor receptor (EGFR) present on various cell surfaces, including human skin. am. It was originally discovered in human urine and was called urogastrone, and is also secreted in various human tissues. During the general skin wound healing process (hemostasis-inflammation-proliferation-maturation stages), the role of epithelial cell growth factors is especially important in the proliferation stage. It proliferates epithelial cells and re-epithelializes the wound area to cover the skin. It stimulates the secretion of different types of growth factors and promotes the proliferation of granulation tissue that fills the new flesh and the creation of blood vessels.

Despite the many known benefits, there are limitations to the industrial and medical use of epidermal growth factor. In order to promote cell growth and regeneration, the binding of growth factors to receptors on the cell surface is essential. If growth factor receptors are overexpressed, excessive cell proliferation can lead to cancer, so epithelial cell growth factors are needed in skin under normal conditions. The number of receptors is limited. Therefore, even if a large amount of epithelial cell growth factors is treated, it is difficult to improve the ability to improve skin damage by increasing cell proliferation.

Therefore, in order to overcome the limitations and problems of existing epithelial cell growth factors, the present inventors produced a multimeric fusion protein through fusion with ferritin protein, and combined zinc to form the enhanced epithelial cell growth factor fusion complex of the present invention. Produced. This enhances cell proliferation and wound healing, so it is expected to be used for skin regeneration or damage prevention, improvement, or treatment.

Korean Patent No. 10-1040168

The present inventors confirmed the effectiveness of the enhanced epidermal growth factor fusion complex for preventing, improving, or treating skin regeneration and damage, and completed the present invention based on this finding.

Accordingly, the object of the present invention is EGF (epithelial growth factor); ferritin; and zinc, to provide a recombinant epithelial cell growth factor fusion complex.

Another object of the present invention is EGF (epithelial growth factor); and a polynucleotide encoding a fusion protein containing ferritin.

Another object of the present invention is (a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;

(b) transforming the vector into a host cell; and

(c) providing a method for producing a recombinant epidermal growth factor fusion protein, including the step of purifying the fusion protein from the host cell.

Another object of the present invention is (a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;

(b) transforming the vector into a host cell;

(c) purifying the fusion protein from the host cells; and

(d) providing a method for producing a recombinant epidermal growth factor fusion complex, comprising the step of binding zinc to the fusion protein.

Another object of the present invention is to provide a pharmaceutical composition, cosmetic composition, food composition, and quasi-drug composition for skin regeneration or for preventing, improving, or treating skin damage, containing the fusion complex of the present invention as an active ingredient. .

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the object of the present invention, the present invention includes EGF (epithelial growth factor); ferritin; and zinc, and provides a recombinant epidermal growth factor fusion complex.

In addition, the present invention provides EGF (epithelial growth factor); and a polynucleotide encoding a fusion protein containing ferritin.

In addition, the present invention provides (a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;

(b) transforming the vector into a host cell; and

It provides a method for producing a recombinant epithelial growth factor fusion protein, comprising the step of (c) purifying the fusion protein from the host cells.

In addition, the present invention provides (a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;

(b) transforming the vector into a host cell;

(c) purifying the fusion protein from the host cells; and

(d) It provides a method for producing a recombinant epidermal growth factor fusion complex, comprising the step of binding zinc to the fusion protein.

Additionally, the present invention provides a pharmaceutical composition for skin regeneration or prevention or treatment of skin damage, comprising the fusion complex of the present invention as an active ingredient.

Additionally, the present invention provides a cosmetic composition for skin regeneration or for preventing or improving skin damage, comprising the fusion complex of the present invention as an active ingredient.

Additionally, the present invention provides a food composition for skin regeneration or for preventing or improving skin damage, comprising the fusion complex of the present invention as an active ingredient.

In addition, the present invention provides a quasi-drug composition for skin regeneration or for preventing or improving skin damage, comprising the fusion complex of the present invention as an active ingredient.

In one embodiment of the present invention, the ferritin may be characterized as ferritin light chain, but is not limited thereto.

In another embodiment of the present invention, the zinc may be characterized as binding to a complex containing EGF and ferritin, but is not limited thereto.

In another embodiment of the present invention, the fusion complex may be characterized as promoting the binding of epithelial cell growth factor and epithelial cell growth factor receptor, but is not limited thereto.

In another embodiment of the present invention, the zinc may bind to the complex containing EGF and ferritin at a ratio of 1:24 to 1:4500, but is not limited thereto.

In another embodiment of the present invention, the fusion complex may be characterized as comprising an amino acid sequence represented by SEQ ID NO: 6, but is not limited thereto.

In another embodiment of the present invention, the polynucleotide may be characterized as comprising base sequences represented by SEQ ID NOs: 1 to 5, but is not limited thereto.

In another embodiment of the present invention, the composition may be characterized as satisfying one or more of the following characteristics, but is not limited thereto: (i) increasing the cell proliferative power of keratinocytes; and (ii) increasing the wound healing ability of keratinocytes.

In addition, the present invention provides a method of skin regeneration or a method of preventing, improving, or treating skin damage, including the step of administering a composition containing the fusion complex as an active ingredient to an individual in need thereof.

In addition, the present invention provides a cosmetic method for skin regeneration or prevention or improvement of skin damage, comprising the step of administering a composition containing the above fusion complex as an active ingredient to an individual in need thereof.

In addition, the present invention provides a use of a composition containing the above fusion complex as an active ingredient for skin regeneration or for preventing, improving, or treating skin damage.

In addition, the present invention provides the use of a composition containing the above fusion complex as an active ingredient for producing cosmetics used for skin regeneration or prevention or improvement of skin damage.

In addition, the present invention provides the use of a composition containing the above fusion complex as an active ingredient for producing a drug used for skin regeneration or prevention or treatment of skin damage.

The reinforced epithelial cell growth factor fusion complex combined with zinc according to the present invention promotes dimerization of the epithelial cell growth factor receptor and effectively supplies zinc into cells, significantly improving the proliferation ability and wound healing ability of keratinocytes. I confirmed what I ordered. Therefore, it is expected that the composition containing the reinforcing epithelial cell growth factor can be used for skin regeneration, prevention, improvement, or treatment of damage.

Figure 1 shows His tag forming a fusion protein; ferritin light chain; It shows a schematic diagram in which synthetic genes encoding both epidermal growth factor (EGF) and epidermal growth factor (EGF) were inserted into the pET28b plasmid, a protein expression vector for E. coli.
Figure 2 shows an electrophoresis image showing the purity of enhanced epithelial growth factor (eEGF) protein. M represents the molecular weight marker, T represents the total E. coli pulverized product, and E represents the final purified enhanced epidermal growth factor (eEGF) fusion protein of 27.5 kDa in size.
Figure 3 shows the cell proliferation ability after treating human keratinocytes with enhanced epithelial growth factor (eEGF) fusion complexes containing PBS, EGF, and zinc.
Figure 4 shows the wound healing ability after treating human keratinocytes with an enhanced epidermal growth factor (eEGF) fusion complex combined with PBS, EGF, and zinc, and an enhanced epidermal growth factor (eEGF) fusion complex without zinc, respectively. It has been confirmed.
Figure 5 is a schematic diagram in which epidermal growth factor, which forms a cluster around a spherical ferritin protein template bound to zinc, binds to an epidermal growth factor receptor dimer.

In one embodiment of the present invention, the cell proliferation ability of the enhanced epidermal growth factor fusion complex was measured, and in the case of zinc-conjugated enhanced epidermal growth factor (eEGF), it was confirmed that the cell proliferation ability was significantly improved compared to the control group ( See Example 4).

In another example of the present invention, the wound healing ability of the enhanced epidermal growth factor fusion complex was measured, and it was confirmed that the wound healing ability of the enhanced epidermal growth factor (eEGF) with or without zinc was significantly improved compared to the control group ( See Example 5).

Therefore, it can be confirmed that the zinc-bound reinforced epithelial cell growth factor fusion complex of the present invention significantly increased the proliferative power of keratinocytes and significantly improved wound healing ability, including the strengthened epithelial cell growth factor. It is expected that the composition can be used for skin regeneration and prevention or treatment of damage.

Hereinafter, the present invention will be described in detail.

The present invention relates to EGF (epithelial growth factor); ferritin; and zinc, and provides a recombinant epidermal growth factor fusion complex.

In the present invention, epidermal growth factor (EGF) promotes cell proliferation and differentiation by binding to the epidermal growth factor receptor (EGFR) present on the surface of various cells, including human skin. It is a protein that promotes In the present invention, the base sequence encoding the epithelial cell growth factor may be represented by SEQ ID NO: 5, but is not limited thereto.

In the present invention, ferritin is an important protein responsible for iron storage in the human body and forms a highly multimer (polymer), thereby helping to form a cluster of epidermal growth factor fused to the C-terminus. . The cluster may be a spherical fusion body containing 24 epithelial cell growth factor monomers, but is not limited thereto.

The ferritin may be a heavy chain or a light chain, and in one embodiment of the present invention, a fusion complex is formed using a light chain, but the present invention is not limited thereto. In the present invention, the ferritin can be replaced with a protein that forms a multimer, such as sHSP (small heat shock protein), and the protein that forms the multimer can form a cluster of epithelial cell growth factors. However, it is not limited to this. Additionally, the base sequence encoding the ferritin may be represented by SEQ ID NO: 3, but is not limited thereto.

In addition, the ferritin can cause epithelial cell growth factors to protrude out of the spherical fusion body and form clusters in which epithelial cell growth factors are concentrated, but is not limited to this.

In the present invention, before forming the fusion complex of the present invention, a protein complex containing the EGF and ferritin may be formed, but is not limited thereto.

In the present invention, zinc is a mineral essential for metabolic processes in cells and tissues. Additionally, zinc is a component of enzymes and is involved in the synthesis and decomposition of carbohydrates, proteins, lipids, and nucleic acids. In particular, it is involved in the synthesis of nucleic acids, DNA and RNA, and plays an essential role in the process of cell differentiation, proliferation, and gene expression, ensuring smooth growth, tissue and skeletal formation, reproduction, and immune functions. In the present invention, the zinc may bind to a complex containing EGF and ferritin, and specifically, may bind to the inside or outside of the complex or may be encapsulated inside the complex, but is not limited thereto. The zinc may be replaced with various metal ions or essential minerals, and specifically, calcium, magnesium, potassium, manganese, iron, copper, iodine, selenium, or chromium, but is not limited thereto.

In the present invention, the zinc may be bound to the complex at a ratio of 1:24 to 1:4500, but is not limited thereto. In addition, the zinc can be bound to the inside and outside of the complex at a ratio of 1:24 to 1:4500, and specifically to the internal nucleation site made of glutamic acid at a ratio of 1:24 to 1:4500. It can be combined in a ratio of, but is not limited to this.

In the present invention, the fusion complex may include the amino acid sequence of SEQ ID NO: 6, or preferably may consist of the amino acid sequence of SEQ ID NO: 6, but is not limited thereto. Additionally, the fusion complex may be formed by fusing ferritin protein and epithelial cell growth factor, and may be a spherical multimeric polymer in which multiple epithelial cell growth factors are gathered together, but is not limited thereto. In addition, the fusion complex can promote the binding of epithelial cell growth factor and epithelial cell growth factor receptor, and can absorb zinc bound to the fusion complex into cells, but is not limited to this. In the present invention, the spherical multimeric polymer in which a plurality of the epidermal growth factors are gathered is 5 to 50, 5 to 40, 5 to 30, 10 to 50, 10 to 40, 10 to 30, and 15. It may include, but is not limited to, 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, 20 to 30, or 24 epithelial cell growth factors. In addition, the fusion complex can bind to the epithelial cell growth factor receptor on the cell surface and be absorbed into cells through an incorporation process, and can promote the movement of skin cells, but is not limited to this. Additionally, the fusion complex can improve wound regeneration and healing ability, but is not limited thereto. Additionally, the fusion complex may promote dimerization of the epithelial cell growth factor receptor, which is essential for the transmission of cell growth signals, but is not limited thereto.

The present invention relates to EGF (epithelial growth factor); and a polynucleotide encoding a fusion protein containing ferritin.

In the present invention, the fusion protein has a His tag; thrombin cleavage enzyme recognition site; Ferritin protein light chain (excluding the N-terminal 13 amino acids); TEV cleavage enzyme recognition site; and a recombinant epidermal growth factor fusion protein containing epidermal growth factor, but is not limited thereto. Additionally, the fusion protein includes functional equivalents of the above proteins. The "functional equivalent" refers to one that has at least 80%, more preferably 90% or more sequence homology to the amino acid sequence as a result of addition, substitution or deletion of amino acids, and has substantially the same physiological activity as the fusion protein. refers to a polypeptide that represents A method of exchanging amino acids in a protein without significantly changing the physiological activity of the protein can be done by methods known in the art.

In the present invention, a “recombinant vector” is a genetic construct containing essential regulatory elements operably linked to express the gene insert, and may be a plasmid vector, cosmid vector, bacteriophage vector, or viral vector. , but is not limited to this. The polynucleotide sequence according to the present invention can be operably linked to an expression control sequence, and the operably linked gene sequence and the expression control sequence include a selection marker and a replication origin. It may be included in an expression vector. “Operably linked” may be a gene and an expression control sequence that are linked in a manner that allows gene expression when an appropriate molecule is linked to the expression control sequence. The “expression control sequence” refers to a DNA sequence that regulates the expression of an operably linked polynucleotide sequence in a specific host cell. Such regulatory sequences include a promoter to effect transcription, optional operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate termination of transcription and translation. Examples of the above plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118, pUC119, pET22b(+), pET28b), Bacillus subtilus-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, and YCp50). The virus may be an animal virus such as a retrovirus, adenovirus, or vaccinia virus, or an insect virus such as a baculovirus. A vector suitable for introducing the polynucleotide according to the present invention into a host cell can be used, and preferably, a vector designed to facilitate induction of protein expression and isolation of the expressed protein can be used. Recombinant vectors containing the polynucleotide of the present invention can be introduced into host cells using methods known in the art. Methods for introducing the recombinant vector according to the present invention into host cells include, but are not limited to, calcium chloride (CaCl ₂ ) and heat shock methods, particle gun bombardment, and silicon carbide. Silicon carbide whiskers, sonication, electroporation, and precipitation with PEG (polyethylenglycol) can be used.

In the present invention, the polynucleotide may include base sequences represented by SEQ ID NOs: 1 to 5, but is not limited thereto. Additionally, the polynucleotide may be single or double stranded with a base sequence, and may be DNA or RNA. The polynucleotide may be isolated from nature or produced by chemical synthesis.

In addition, the vector has a base sequence having at least 90%, more preferably at least 95%, and most preferably at least 98% sequence homology to the base sequence represented by SEQ ID NO: 1, 2, 3, 4, or 5. may include. The “% sequence homology” is determined by comparing an optimally aligned sequence with a comparison region, wherein a portion of the polynucleotide sequence is compared to a reference sequence (without additions or deletions) for the optimal alignment of the sequences. May contain additions or deletions (i.e. gaps).

The present invention provides (a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;

(b) transforming the vector into a host cell; and

It provides a method for producing a recombinant epithelial growth factor fusion protein, comprising the step of (c) purifying the fusion protein from the host cells.

In addition, the present invention provides (a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;

(b) transforming the vector into a host cell;

(c) purifying the fusion protein from the host cells; and

(d) It provides a method for producing a recombinant epidermal growth factor fusion complex, comprising the step of binding zinc to the fusion protein.

In the present invention, the host cell is preferably bacteria, more preferably Escherichia coli, but is not limited thereto.

In addition, the separation and purification of the fusion protein can be performed through various separation and purification methods known in the art, for example, after lysing the cells, centrifuging the lysate, salting out (ammonium sulfate precipitation and Techniques such as sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange chromatography, reversed phase column chromatography, and affinity chromatography can be applied alone or in combination. , but is not limited to this.

The present invention provides a pharmaceutical composition for skin regeneration or prevention or treatment of skin damage, comprising the fusion complex of the present invention as an active ingredient.

The composition may be characterized as satisfying one or more of the following characteristics, but is not limited thereto: (i) increasing the cell proliferative power of keratinocytes; and (ii) increasing the wound healing ability of keratinocytes. The wound healing ability may mean recovery of skin wounds through promotion of cell proliferation, but is not limited thereto.

The content of the fusion complex in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight, based on the total weight of the composition. It may be, but is not limited to this. The content ratio is a value based on the dry amount with the solvent removed.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, paste preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

The present invention provides a cosmetic composition for skin regeneration or preventing or improving skin damage, comprising the fusion complex of the present invention as an active ingredient.

The formulation of the cosmetic composition according to the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, mist, moisture cream, hand cream, hand lotion, foundation, It may be in the form of essence, nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, cleansing oil, cleansing balm, body lotion, or body cleanser.

The cosmetic composition of the present invention may further include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, and sphingolipids.

Water-soluble vitamins may be any that can be mixed into cosmetics, but examples include vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, and vitamin H. and their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate, etc.) are also water-soluble vitamins that can be used in the present invention. Included. Water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial cultures, enzymatic methods, or chemical synthesis.

Oil-soluble vitamins may be any that can be mixed into cosmetics. Examples include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol), and their derivatives (palmitic acid). Ascorbic acid, ascorbic acid stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetic acid, dl-alpha tocopherol nicotinic acid, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl ether, etc.) Included in the oil-soluble vitamins used in the invention. Oil-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial cultures, enzymatic or chemical synthesis.

Any polymer peptide that can be blended into cosmetics may be used, and examples include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. High molecular weight peptides can be purified and obtained by conventional methods such as purification from microbial culture media, enzymatic methods, or chemical synthesis, or they can usually be purified and used from natural products such as dermis of pigs or cows or silk fiber of silkworms.

The high molecular weight polysaccharide may be anything that can be blended into cosmetics, but examples include hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate, or its salts (sodium salt, etc.). For example, chondroitin sulfate or its salt can usually be purified and used from mammals or fish.

The sphingolipid may be any one that can be blended into cosmetics, and examples include ceramide, phytosphingosine, and sphingolipid. Sphingolipids can usually be purified by conventional methods from mammals, fish, shellfish, yeast, or plants, or obtained by chemical synthesis.

In addition to the above-mentioned essential ingredients, the cosmetic composition of the present invention may contain other ingredients usually blended in cosmetics as needed.

Other ingredients that may be added include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, colorants, fragrances, Examples include blood circulation promoters, cooling agents, antiperspirants, and purified water.

Examples of oil and fat components include ester-based fats and oils, hydrocarbon-based fats and oils, silicone-based fats and oils, fluorine-based fats and oils, animal fats and oils, and vegetable fats and oils.

Ester oils include glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, and stearic acid. Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, adipine. Diisopropyl acid, isoalkyl neopentanoate, tri(caprylic, capric acid)glyceryl, trimethylolpropane tri2-ethylhexanoate, trimethylolpropane triisostearate, pentaelislitol tetra2-ethylhexanoate , cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinooleate, isostear laurate. Reyl, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, cetoester 2-ethylhexanoate Aryl, 2-ethylhexanoate stearyl, hexyl isostearate, ethylene glycol dioctanate, ethylene glycol dioleate, propylene glycol dicapric acid, di(capryl, capric acid) propylene glycol, propylene glycol dicaprylate, dicapric acid Neopentyl glycol prinate, neopentyl glycol dioctanate, glyceryl tricaprylate, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate. , octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleic acid ester, polyglycerol isostearic acid ester, Triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyltriethyl citrate, acetyltributyl citrate, trioctyl citrate, dimalate Isostearyl, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate, Cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytsteryl isostearate, phytsteryl oleate, 12-stealoyl hydroxystearate isocetyl, 12-stealoyl hydride. Ester systems such as stearyl hydroxystearate and isostearyl 12-stealoylhydroxystearate can be mentioned.

Examples of hydrocarbon oils include squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybudene, microcrystalline wax, and petroleum jelly.

Silicone oils include polymethyl silicone, methylphenyl silicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane/methylcetyloxysiloxane copolymer, dimethylsiloxane/methylstealoxysiloxane copolymer, and alkyl. Modified silicone oil, amino-modified silicone oil, etc. can be mentioned.

Examples of fluorine-based fats and oils include perfluoropolyether.

Animal or plant oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, birdflower oil, soybean oil, corn oil, rapeseed oil, apricot oil, palm kernel oil, palm oil, castor oil, sunflower oil, and grape seed oil. , cottonseed oil, palm oil, cucumber nut oil, wheat germ oil, rice germ oil, shea butter, walnut colostrum oil, marker damia nut oil, meadow oil, egg yolk oil, beef tallow, horse oil, mink oil, orange rape oil, jojoba oil. , animal or plant oils such as candelier wax, carnaba wax, liquid lanolin, and hydrogenated castor oil.

Moisturizers include water-soluble low-molecular-weight moisturizers, oil-soluble molecular moisturizers, water-soluble polymers, and fat-soluble polymers.

Water-soluble low-molecular-weight moisturizers include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (degree of polymerization n=2 or more), and polypropylene. Examples include glycol (degree of polymerization n=2 or more), polyglycerin B (degree of polymerization n=2 or more), lactic acid, and lactate salts.

Examples of fat-soluble low-molecular-weight moisturizers include cholesterol and cholesterol esters.

Water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water-soluble chitin, chitosan, and dextrin. You can.

Examples of oil-soluble polymers include polyvinylpyrrolidone/eicosene copolymer, polyvinylpyrrolidone/hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

Examples of emollients include long-chain acyl glutamic acid cholesteryl ester, hydroxystearic acid cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid, and lanolin fatty acid cholesteryl ester.

Examples of surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE. Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE/POP (polyoxyethylene/polyoxypropylene) copolymer, POE/POP alkyl ether, polyether modified silicone, lauric acid. Examples include alkanolamide, alkylamine oxide, and hydrogenated soybean phospholipid.

Anionic surfactants include fatty acid soap, alpha-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkyl amide sulfate, alkyl phosphate, POE alkyl phosphate, and alkyl amide phosphate. , alkyloyl alkyl taurine salt, N-acylamino acid salt, POE alkyl ether carboxylate, alkyl sulfosuccinate, sodium alkyl sulfoacetate, acylated hydrolyzed collagen peptide salt, perfluoroalkyl phosphate ester, etc. .

Cationic surfactants include alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyltrimethylammonium bromide, and cationic surfactant. Benzalkonium, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, quaternary ammonium salt of lanolin derivative, etc. are mentioned.

Amphoteric surfactants include carboxy beta type, amide beta type, sulfo beta type, hydroxy sulfo beta type, amide sulfo beta type, phosphobeta type, aminocarboxylate type, imidazoline derivative type, and amide amine type. Amphoteric surfactants, etc. can be mentioned.

Organic and inorganic pigments include silicic acid, anhydrous silicic acid, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium-coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, and aluminum oxide. Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine and complexes thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene/styrene copolymer, Examples include organic pigments such as silk powder, cellulose, CI pigment yellow, and CI pigment orange, and complex pigments of these inorganic pigments and organic pigments.

Examples of organic powder include metal soap such as calcium stearate; Alkyl phosphate metal salts such as sodium zinc cetilate, zinc laurylate, and calcium laurylate; Acyl amino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroyl glycine calcium; Amidesulfonic acid polyvalent metal salts such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylizine, N-alpha-paritoylolnithine, N-alpha-lauroylarginine, N-alpha-hydrogenated beef tallow fatty acid acylarginine, etc. -Acyl basic amino acid; N-acyl polypeptides such as N-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Examples include polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene/styrene copolymer, and ethylene tetrafluoride.

UV absorbers include para-aminobenzoic acid, ethyl para-aminobenzoate, amyl para-aminobenzoate, octyl para-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, and benzyl cinnamate. , paramethoxycinnamic acid-2-ethoxyethyl, paramethoxycinnamic acid octyl, diparamethoxycinnamic acid mono-2-ethylhexane glyceryl, paramethoxycinnamic acid isopropyl, diisopropylㆍdiisopropylcinnamic acid ester mixture, uro Kaninic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone disulfonate sodium, dihydroxybenzophenone , tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1'-oxy)-1 , 3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc.

Disinfectants include hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zincphyllithione, benzalkonium chloride, and photosensitive. Sub-No. 301, mononitroguaiacol sodium, undecirenic acid, etc. can be mentioned.

Antioxidants include butylhydroxyanisole, propyl gallate, and elisorbic acid.

Examples of pH adjusters include citric acid, sodium citrate, malic acid, sodium malate, fumal acid, sodium fumalate, succinic acid, sodium succinate, sodium hydroxide, and sodium monohydrogen phosphate.

Examples of alcohol include higher alcohols such as cetyl alcohol.

In addition, the mixing ingredients that may be added are not limited to this, and any of the above ingredients can be mixed within a range that does not impair the purpose and effect of the present invention, but in an amount of 0.01-5% by weight or 0.01-3% based on the total weight. Can be formulated in % weight percentages.

When the formulation of the present invention is a lotion, paste, cream or gel, the carrier ingredients include animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. It can be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide ether. Sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linoline derivatives, or ethoxylated glycerol fatty acid esters can be used.

In another aspect of the present invention, the present invention provides a food composition for skin regeneration or for preventing or improving skin damage, comprising the fusion complex of the present invention as an active ingredient.

In the present invention, “food” means a natural product or processed product containing one or more nutrients, preferably in a state that can be eaten directly after a certain degree of processing, and has the usual meaning. This means that it includes all health functional foods, beverages, food additives, and beverage additives.

In the present invention, the food composition may be a health functional food composition, but is not limited thereto.

In the present invention, the “functional food” is the same term as food for special health use (FoSHU), and is a medical product processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. It refers to a food with high medical effectiveness and can be manufactured in tablet, capsule, pill, granule, powder, liquid, flake, paste, syrup, gel, jelly, bar, or film formulations. Here, “functionality” means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects.

When using the fusion complex of the present invention as a food additive, it can be added as is or used with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the fusion complex of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, These include alcoholic beverages and vitamin complexes, and include all health functional foods in the conventional sense.

The health drink composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of natural carbohydrates is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain carbonating agents used in carbonated drinks. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

The health functional food can have the advantage of having better effects when consumed in the form of inner beauty food. The inner beauty is a food called 'edible cosmetics or beauty food'. It refers to food that changes the skin constitution to a healthy one by absorbing various ingredients that are good for the skin into the body. It refers to the food that changes the skin constitution to a healthy one by selecting cosmetics that suit your skin type. You can choose and consume inner beauty foods that suit each individual, considering their skin condition and lifestyle. For example, when using a combination of cosmetics containing the cosmetic composition and inner beauty food containing the fusion complex, the effect is significantly higher compared to using only cosmetics or drugs, resulting in more effective skin regeneration or skin damage prevention and improvement effects. You can have the advantage of being able to see it.

In addition, the present invention provides a quasi-drug composition for skin regeneration or for preventing or improving skin damage, comprising the fusion complex of the present invention as an active ingredient.

In the present invention, “quasi-drugs” refer to products with a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act of the Republic of Korea. According to it, quasi-drugs exclude products used for medicinal purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.

Additionally, the quasi-drugs may include external skin preparations and personal hygiene products. For example, it may be a disinfectant cleanser, shower foam, garnish, wet tissue, detergent soap, hand wash, body cleanser, soap, mask, cream, lotion, essence, spray, or ointment, but is not limited thereto.

When using the quasi-drug composition according to the present invention as a quasi-drug additive, the composition can be added as is or used together with other quasi-drugs or quasi-drug components, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

For example, the quasi-drug composition of the present invention may be manufactured in the form of a general emulsified formulation or solubilized formulation. For example, it may have a formulation such as lotion, cream, ointment, spray, oil gel, gel, oil, aerosol, or smoke screen, but can be used without limitation as long as it exhibits the pest control inducing effect of the present invention. . In addition, the above-mentioned quasi-drug compositions are appropriately mixed with oil, water, surfactants, moisturizers, lower alcohols with 1 to 4 carbon atoms, thickeners, chelating agents, colorants, preservatives or fragrances, etc., which are generally mixed in quasi-drug compositions, in each dosage form as needed. You can use it.

In the present invention, “prevention” refers to all actions that suppress or delay the onset of the desired disease, and “treatment” refers to the prevention of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It means any action that improves or changes beneficially, and “improvement” means any action that reduces parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

In the present invention, “individual” means a subject in need of treatment for a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, and Refers to mammals such as cows.

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

Below, preferred examples and experimental examples are presented to aid understanding of the present invention. However, the following examples and experimental examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Gene synthesis and vector construction of enhanced epithelial growth factor

A DNA construct containing sequences 3, 4, and 5 of Table 1 was synthesized by requesting Bionics Co., Ltd., and the DNA construct was again synthesized by Bionics Co., Ltd. using NdeI and XhoI of the pET28b plasmid for expression in E. coli. By cloning through restriction enzyme sites, a vector for expressing the enhanced epidermal growth factor fusion protein containing SEQ ID NOs. 1 to 5 shown in Table 1 was finally created. This is shown in Figure 1. Additionally, Table 1 below shows the base sequences used in the present invention.

gene Base sequence (5'-> 3') sequence number His tag CATCATCATCATCATCAC One thrombin site AGCAGCGGCTGGTGCCGCGCGGCAGC 2 ferritin light chain CATATGGAGGCAGCCGTCAACAGCCTGGTCAATTT
GTACCTGCAGGCCTCCTACACCTACCTCTCTCTGG
GCTTCTATTTCGACCGCGATGATGTGGCTCTGGAA
GGCTGAGCCACTTCTTCCGCGAACTGGCCGAGGA
GAAGCGCGAGGGCTACGAGCGTCTCCTGAAGATGC
AAAACCAGCGTGGGCGGCCGCGCTCTCTTCCAGGAC
ATCAAGAAGCCAGCTGAAGATGAGTGGGGTAAAAC
CCCAGACGCCATGAAAGCTGCCATGGCCCTGGAGA
AAAAGCTGAACCAGGCCCTTTTGGATCTTCATGCC
CTGGGTTCTGCCCGCACGGACCCCCATCTCTGTGA
CTTCCTGGAGACTCACTTCCTAGATGAGGAAGTGA
AACTGATCAAGAAGATGGGTGACCACCTTGACCAAC
CTCCACAGGCTGGGTGGCCCGGAGGCTGGGCTGGG
CGAGTATCTCTTCGAAAGGCTCACTCTCAAGCACGAC 3 TEV site GAAAACCTGTATTTTCAGGGA 4 epidermal growth factor AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAA
GCATTGGACAAGTATGCATGCAACTGTGTTGTTGGC
TACATCGGGGAGCGATGTCAGTACCGAGACCTGAAG
TGGTGGGAACTGCGCTGA 5

Example 2. Production and cultivation of transformed strains into which enhanced epithelial cell growth factors were introduced.

The prepared vector for expressing the enhanced epidermal growth factor fusion protein was introduced into E. coli BL21 (DE3) strain by heat shock method, spread on Luria Bertani solid medium containing 30 mcg/ml kanamycin, and cultured overnight in an incubator at 37°C. did. The next day, single colonies were selected and inoculated again into Luria Bertani liquid medium. After culturing for several hours, a small amount was taken, 20% glycerol was added, and stored frozen as cell stock at -80°C.

Additionally, the frozen transformed strain was inoculated into Luria Bertani liquid medium containing 30 mcg/ml of kanamycin and cultured in a shaking incubator set at 37°C until the absorbance OD ₆₀₀ reached 0.5. Overexpression of the fusion protein was induced by adding 1mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) to the culture medium, and then cultured for an additional 16 hours, and then the strain was recovered using a centrifuge.

Example 3. Purification and fusion complex production of enhanced epithelial cell growth factor fusion protein

A buffer solution consisting of 50mM Tris pH 7.5/5mM EDTA/2% triton x-100 was added to the recovered transformed E. coli strain, and pulverized using an ultrasonicator. Then, the mixture was centrifuged at 8,000 rpm for 15 minutes using a centrifuge to separate the inclusion body containing the overexpressed epidermal growth factor fusion protein. The isolated inclusion body was released by denaturing the recombinant fusion protein with a buffer solution consisting of 50 mM glycine pH9.0/0.1 M NaCl/8 M urea and then flowed through a nickel-chelating column (GE healthcare). The fusion protein bound in the column was eluted with a buffer solution containing 50 mM glycine pH9.0/0.1 M NaCl/8 M urea/0.5 M imidazole, and purity was confirmed using electrophoresis (SDS-PAGE). The results are shown in Figure 2. Additionally, the amino acid sequence of the epidermal growth factor fusion protein is shown in Table 2 below.

As shown in Figure 2, the final purified epidermal growth factor fusion protein was confirmed to be 27.5 kDa.

In addition, the eluted recombinant epidermal growth factor fusion protein was treated with 10mM DTT for 4 hours to remove disulfide bonds, and then washed with a buffer solution consisting of 50mM glycine pH9.0/0.1M NaCl/10% glycerol through dialysis. Refolding of the fusion protein was performed within. The refolded recombinant epidermal growth factor fusion protein was treated with 1mM ZnSO ₄ for 1 hour and then desalted using a desalting column (GE healthcare) to finally complete a pure recombinant enhanced epidermal growth factor (eEGF) fusion complex.

protein amino acid sequence sequence number Epidermal growth factor fusion protein MGSSHHHHHHSSGLVPRGSHMEAAVNSLVNLYLQASYTYLSLGFYFDRDD
VALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGK
TPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLI
KKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHDENLYFQGNSDSECPLSH
DGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR 6

Example 4. Confirmation of the effect of increasing cell proliferation of recombinant enhanced epithelial cell growth factor

To measure the cell proliferation ability of the enhanced epithelial cell growth factor fusion complex of the present invention, HaCaT cells, which are human keratinocytes, were cultured using DMEM high glucose medium (Welgene) containing 10% FBS, and then cultured in a 24-well plate. 7 × 10 ⁴ cells were distributed into three groups, each well, into three groups. The next day, the medium in the 24-well plate was replaced with serum-free DMEM high glucose medium (Welgene), and after 16 hours, the first group (negative control) was administered PBS and the second group (positive control) was administered 1 mcg/ml. EGF (Sigma-Aldrich) was administered, and the third group (treatment group) was treated with 1 mcg/ml zinc-conjugated enhanced epidermal growth factor (eEGF) fusion complex. After 24 hours, the HaCaT cells proliferated in each well were quantitatively compared using the MTT assay. The results are shown in Figure 3.

As shown in Figure 3, in the case of zinc-conjugated enhanced epidermal growth factor (eEGF), cell proliferation was confirmed to be significantly improved compared to the control group.

Example 5. Confirmation of the effect of increasing the wound healing ability of recombinant enhanced epithelial cell growth factor

To measure the wound healing ability of the enhanced epidermal growth factor fusion complex of the present invention, HaCaT cells, which are human keratinocytes, were cultured using DMEM high glucose medium (Welgene) containing 10% FBS, and then cultured in a 6-well plate. 3.5×10 ⁵ cells each were dispensed into four wells. The next day, the medium in the 6-well plate was replaced with serum-free DMEM high glucose medium (Welgene), and after 16 hours, the center of each well was scraped to a certain thickness, and then PBS was added to the first well (negative control) and the second well. One well (positive control) was administered 1 mcg/ml of EGF (Sigma-Aldrich), the third well (treatment group) was administered 1 mcg/ml of zinc-conjugated enhanced epidermal growth factor fusion complex, and the fourth well (treatment group) was administered 1 mcg/ml of zinc-conjugated epidermal growth factor fusion complex. Treatment group) was treated with 1 mcg/ml of enhanced epidermal growth factor fusion complex without zinc. After 24 hours, the area filled with scratched HaCaT cells was compared using the scratch assay method. The results are shown in Figure 4.

As shown in Figure 4, it was confirmed that the wound healing ability of zinc-bound enhanced epidermal growth factor (eEGF) was significantly improved compared to the control group.

In summary, it can be seen that the reinforced epithelial cell growth factor fusion complex combined with zinc significantly increased the proliferative power of keratinocytes and significantly improved the wound healing ability, and the composition containing the strengthened epithelial cell growth factor It is expected that it can be used for skin regeneration and prevention or treatment of damage.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> A complex with epidermal growth factor and use thereof <130> MP21-253 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> His tag <400> 1 catcatcatc atcatcac 18 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> thrombin site <400> 2 agcagcggcc tggtgccgcg cggcagc 27 <210> 3 <211> 492 <212> DNA <213> Artificial Sequence <220> <223> ferritin light chain <400> 3 catatggagg cagccgtcaa cagcctggtc aatttgtacc tgcaggcctc ctacacctac 60 ctctctctgg gcttctattt cgaccgcgat gatgtggctc tggaaggcgt gagccacttc 120 ttccgcgaac tggccgagga gaagcgcgag ggctacgagc gtctcctgaa gatgcaaaac 180 cagcgtggcg gccgcgctct cttccaggac atcaagaagc cagctgaaga tgagtggggt 240 aaaaccccag acgccatgaa agctgccatg gccctggaga aaaagctgaa ccaggccctt 300 ttggatcttc atgccctggg ttctgcccgc acggaccccc atctctgtga cttcctggag 360 actcacttcc tagatgagga agtgaaactg atcaagaaga tgggtgacca cctgaccaac 420 ctccacaggc tgggtggccc ggaggctggg ctgggcgagt atctcttcga aaggctcact 480 ctcaagcacg ac 492 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TEV site <400> 4 gaaaacctgt attttcaggg a 21 <210> 5 <211> 162 <212> DNA <213> Artificial Sequence <220> <223> epidermal growth factor <400> 5 aatagtgact ctgaatgtcc cctgtcccac gatgggtact gcctccatga tggtgtgtgc 60 atgtatattg aagcattgga caagtatgca tgcaactgtg ttgttggcta catcggggag 120 cgatgtcagt accgagacct gaagtggtgg gaactgcgct ga 162 <210> 6 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> epidermal growth factor fusion protein <400> 6 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Glu Ala Ala Val Asn Ser Leu Val Asn Leu Tyr 20 25 30 Leu Gln Ala Ser Tyr Thr Tyr Leu Ser Leu Gly Phe Tyr Phe Asp Arg 35 40 45 Asp Asp Val Ala Leu Glu Gly Val Ser His Phe Phe Arg Glu Leu Ala 50 55 60 Glu Glu Lys Arg Glu Gly Tyr Glu Arg Leu Leu Lys Met Gln Asn Gln 65 70 75 80 Arg Gly Gly Arg Ala Leu Phe Gln Asp Ile Lys Lys Pro Ala Glu Asp 85 90 95 Glu Trp Gly Lys Thr Pro Asp Ala Met Lys Ala Ala Met Ala Leu Glu 100 105 110 Lys Lys Leu Asn Gln Ala Leu Leu Asp Leu His Ala Leu Gly Ser Ala 115 120 125 Arg Thr Asp Pro His Leu Cys Asp Phe Leu Glu Thr His Phe Leu Asp 130 135 140 Glu Glu Val Lys Leu Ile Lys Lys Met Gly Asp His Leu Thr Asn Leu 145 150 155 160 His Arg Leu Gly Gly Pro Glu Ala Gly Leu Gly Glu Tyr Leu Phe Glu 165 170 175 Arg Leu Thr Leu Lys His Asp Glu Asn Leu Tyr Phe Gln Gly Asn Ser 180 185 190 Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly 195 200 205 Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val 210 215 220 Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp 225 230 235 240 Glu Leu Arg

Claims (15)

EGF (epithelial growth factor); ferritin; And a recombinant epidermal growth factor fusion complex containing zinc.
According to paragraph 1,
The ferritin is a recombinant epidermal growth factor fusion complex, characterized in that the ferritin light chain.
According to paragraph 1,
The zinc is a recombinant epithelial growth factor fusion complex, characterized in that it binds to a complex containing EGF and ferritin.
According to paragraph 1,
The fusion complex is a recombinant epithelial cell growth factor fusion complex, characterized in that it promotes the binding of epithelial cell growth factor and epithelial cell growth factor receptor.
According to paragraph 3,
The zinc is a recombinant epidermal growth factor fusion complex, characterized in that it binds to the complex containing the EGF and ferritin at a ratio of 1:24 to 1:4500.
According to paragraph 1,
The fusion complex is a recombinant epidermal growth factor fusion complex, characterized in that it contains the amino acid sequence represented by SEQ ID NO: 6.
EGF (epithelial growth factor); And a recombinant vector comprising a polynucleotide encoding a fusion protein containing ferritin.
In clause 7,
The polynucleotide is a recombinant vector, characterized in that it contains the base sequences represented by SEQ ID NOs: 1 to 5.
(a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;
(b) transforming the vector into a host cell; and
(c) A method for producing a recombinant epithelial growth factor fusion protein, comprising the step of purifying the fusion protein from the host cells.
(a) EGF (epithelial growth factor); and constructing an expression vector encoding a fusion protein containing ferritin;
(b) transforming the vector into a host cell;
(c) purifying the fusion protein from the host cells; and
(d) A method for producing a recombinant epithelial growth factor fusion complex, comprising the step of binding zinc to the fusion protein.
A pharmaceutical composition for skin regeneration or prevention or treatment of skin damage, comprising the fusion complex of claim 1 as an active ingredient.
According to clause 11,
A pharmaceutical composition for skin regeneration or prevention or treatment of skin damage, characterized in that the composition satisfies any one or more of the following characteristics:
(i) Increases cell proliferation of keratinocytes; and
(ii) Increases the wound healing ability of keratinocytes.
A cosmetic composition for skin regeneration or preventing or improving skin damage, comprising the fusion complex of claim 1 as an active ingredient.
A food composition for skin regeneration or for preventing or improving skin damage, comprising the fusion complex of claim 1 as an active ingredient.
A quasi-drug composition for skin regeneration or for preventing or improving skin damage, comprising the fusion complex of claim 1 as an active ingredient.