KR20190126619A - Anti-wrinkle composition comprising functional peptides and fibroblast conditioned medium - Google Patents

Abstract

Disclosed is a cosmetic composition comprising, as an active ingredient, a functional peptide and an animal cell conditioned medium capable of effectively improving or preventing skin aging and wrinkles. The present invention provides a cosmetic composition for reducing wrinkles, comprising a fibroblast conditioned medium derived from cultured homogeneous foreskin, hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2, and acetyl tetrapeptide-1, as an active ingredient.

Description

Anti-wrinkle composition comprising functional peptides and fibroblast conditioned medium

The present invention relates to an anti-aging and anti-wrinkle composition comprising a functional peptide and conditioned medium of fibroblasts, and more particularly, inhibits the activity of collagenase, promotes the production of collagen in cells and promotes the proliferation of skin cells. It relates to a functional cosmetic composition exhibiting an promoting effect.

One of the biggest areas of interest in cosmetics is wrinkles, pigmentation, decreased elasticity, dry skin, hair loss, and lack of glossiness associated with aging. The skin undergoes various changes through aging. First, the components of the skin, such as the epidermis, dermis and subcutaneous tissue, become thinner and the extracellular matrix (ECM), which gives elasticity to the skin, changes, and collagen, which accounts for 70-80% of the extracellular matrix, is changed. As the body ages, its production decreases rapidly and is closely related to wrinkle formation, and collagen, elastin, proteoglycans, and glucose aminoglycans, which form the skin connective tissue, ), Laminin, fibronectin, etc., are oxidized and lose their function, and the skin loses its elasticity and wrinkles are excessively formed, thereby changing to aged skin.

The connective tissue fibers of the extracellular matrix include glue fibers (collagen fibers, collagen fibers), reticulated fibers and elastic fibers (elastic fibers, elastic fibers), of which collagen accounts for about 70% of skin connective tissue. Is mostly formed in the fibroblasts of the skin. As we age, collagen content in skin connective tissues decreases due to the degradation and degradation of collagen synthesis. Therefore, the degradation of collagen biosynthesis and the promotion of collagen decomposition may be said to be the biggest cause of skin wrinkle formation.

Collagen biosynthesis processes are controlled by many factors involved in transcription and post-translation levels, and collagen breakdown is the collagenase that breaks down collagen by UV light. The expression of matrix metalloproteases (MMPs) such as Collagenase promotes collagen breakdown, resulting in reduced collagen content. In addition, as the deformation of collagen is accelerated by the external environment, the skin becomes wrinkled and deepened. As a result, skin aging is manifested by cell homogenization, loss of elastin, destruction of collagen, reduction and delay in collagen synthesis. Thus, skin aging occurs in the skin epidermis, but rather in the dermis.

Various cosmetic compositions have been studied to solve the problems of skin aging, and the wrinkle improvement effect of the skin has shown visible results in some. For example, there are various reports of clinical results of skin wrinkle removal for retinoids, especially retinol, which are widely used to improve skin wrinkles, blemishes and pigmentation, and cosmetics containing retinol have wrinkles formed by sunlight. It also effectively improves skin sagging and elasticity. Retinol has been continuously reported on its effectiveness, its unique skin irritation problems and stability problems, which are listed as a wrinkle functional notification material, and are used in expensive products at high prices and are difficult to use as universal materials. Part exists.

Peptide is a substance produced by amide bonds of amino acids. It is a core foundation used in a wide range of fields such as basic clinical research, pharmaceuticals (diagnostic reagents, hormones, antibiotics), and household goods (food additives, antifungals, cosmetics). It is material. In particular, the development of diagnostic, prophylactic, and therapeutic drugs using peptides for diseases caused by various viruses, including hepatitis and AIDS, can lower costs than those using conventional expensive proteins, and provide higher sensitivity, selective specificity, and efficiency than proteins. It is a creature material to be able to do. In addition, since the peptide is synthesized as necessary and naturally disappears when its role is completed, when the peptide is used as a medicine or cosmetics, it is known that there are almost no side effects and excellent action.

The first application of these peptides to cosmetics has been attempted in France since the early 2000s and is currently used worldwide, with high market growth rates. Lipotec, Inc., Spain Sederma, Inc. of France and Pentapharm, Inc. of Switzerland.

Peptides have proven their safety and skin penetration by clinical tests, and their active ingredients promote the proliferation of keratinocytes and fibroblasts, which are the main cells of the skin, and promote the production of intracellular substances such as collagen, elastin, hyaluronic acid, laminin and fibronectin. By accelerating, it is known to activate skin cells as a whole and to help regenerate aged skin.

The advantages of the peptide material for cosmetics are stability and safety. For example, retinol, which is an anti-wrinkle ingredient, is weak and unstable in light and heat, while azireline, which is a peptide preparation, can be used even during the day because there is no such restriction. Even if a large amount is used as a substance, there are no side effects, so it can be used safely even in sensitive eyes. Functional peptides are various ligands necessary for skin care such as cell proliferation, cell migration, inflammatory reaction, angiogenesis and skin pigmentation depending on the diversity of amino acid sequences. It is useful in modulating receptor binding, and is a biologically derived material, and thus it is immunotolerant, and has been used as a major cosmetic material having advantages of high transdermal permeability and ex vivo discharge, and easy synthesis and modification. Compared with conventional protein materials, the efficacy is similar, but the size is so small that it has excellent skin permeability, strong competitiveness in terms of economic production such as purity and price, etc. It is a material equipped with.

Growth factors are typically polypeptides that exhibit various biological effects, some growth factor families that have been found useful for wound healing and / or epidermal remodeling, such as growth factor β (TGF-β), epidermal growth factor (EGF) ), Insulin-like growth factors (IGF-I and IGF-II), iron-derived growth factors (PDGF), and fibroblast growth factors (FGFs).

Among the various growth factors, the most representative and widely used raw material is EGF (Epidermal Growth Factor), which uses a biotechnology called genetic recombination technology, and the US biologist Stanley Cohen It was discovered and won the Nobel Prize in Physiology in 1986. It is a natural skin regeneration material that exists in our body such as saliva, sweat and blood, and is known as an ingredient that plays an excellent role in removing and removing fine lines, maintaining skin elasticity, and regenerating skin as well as growing and regenerating our skin cells.

Recently, a mechanism for male hair loss has been elucidated by a domestic research team.Dickkopf-1 (DKK-1), a male protein-induced 'Wnt' inhibitor, causes male hair loss and is a signaling protein for hair development and growth. It is well known that Wnt plays an important role, but in whiskers, male hormones increase expression of 'insulin-like growth factor (IGF-1)' rather than DKK-1, whereas hair loss is increased. In the ongoing hair, male hormones are known to induce the production of DKK-1 and cause hair cell death, and IGF-1 is being actively researched as a new alternative to hair loss.

As mentioned above, various growth factors besides EGF and IGF-1 are being researched and used as the core raw materials of medical cosmetics, and it is expected that the market ripple and growth potential are great. In addition, research on improving price and stability, which is a weak point of these growth factors, is being actively conducted, and the research is expected to be expanded.

Stem cell cultures are used to produce cosmetics containing culture fluids or extracts, such as peptides and enzymes secreted in stem cell culture, rather than regenerating tissue in medical cosmetics, and are effective in preventing skin aging, improving wrinkles, and whitening. These ingredients come from the stem cell culture process, and the exact name of the ingredients that can be contained in cosmetics is 'human cord blood stem cell culture solution', which is a legal ingredient approved by the KFDA and is a safe ingredient listed in INCI. Stem cell cultures contain a lot of effective growth factors for the skin, and when applied to cosmetics, it is used to help improve skin elasticity and transparency.

Living cells secrete proteins and peptides extracellularly, including growth factors in nutrient media during artificial culture. A medium in which growth factors are secreted through artificial culture of cultured cells is called a 'conditioned medium' (see US Pat. No. 6,372,494), and a growth medium rich in growth factors is a three-dimensional substrate and tissue specific cells ( That is, it is suitable for culturing cell cultures comprising multiple layers of a three-dimensional culture system) and can be advantageously used to prepare functional cosmetic compositions. US Pat. No. 6,372,494, etc. confirms that the complex three-dimensional culture system has many advantages, such as having a larger surface area in a simple two-dimensional culture system, and is utilized for various experiments, wherein the condition medium is a three-dimensional culture system. You can see that it is suitable for building.

Thus, the use of growth factors has gained much acceptance among skin care professionals and is more effectively used to treat skin trauma, to treat skin damage, to prevent wrinkles and other defects.

In conclusion, in order to effectively prevent or improve skin aging and wrinkles, various cosmetic materials have been developed and utilized, and it is necessary to develop new wrinkle improvement compositions using raw materials with proven efficacy. The verification of efficacy is identified as a molecular biological mechanism. It is important to verify with the target clinical trial.

Therefore, the present invention has been made to solve the above problems, to provide a cosmetic composition comprising a functional medium and a fibroblast conditioned medium that can effectively prevent or improve skin aging and wrinkles as an active ingredient.

In order to solve the above problem, the present invention, the conditioned medium and hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl of fibroblasts derived from cultured homogeneous foreskin It provides a cosmetic composition for improving wrinkles comprising tetrapeptide-1 as an active ingredient.

In addition, the fibroblasts provide a cosmetic composition, characterized in that derived from the cell line designated by ATCC Accession No. PTA-11680 or the cell line designated by PTA-11681.

In addition, the hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl tetrapeptide-1 with respect to the total composition are included in 0.005 to 0.1% (w / v), respectively, The condition medium provides a cosmetic composition, characterized in that contained in 1 to 10% (v / v).

In addition, the cosmetic composition provides a cosmetic composition, characterized in that to promote the growth of skin fibroblasts.

In addition, the cosmetic composition provides a cosmetic composition, characterized in that to inhibit the activity of MMP-1.

In addition, the cosmetic composition provides a cosmetic composition, characterized in that to promote the production of collagen in the cell.

In addition, the cosmetic composition is a cosmetic composition characterized in that it has a flexible cosmetics, nourishing cosmetics, eye cream, nutrition essence, nutrition cream, massage cream, body cream, cleansing cream, cleansing foam, cleansing water, powder or pack formulation to provide.

According to the present invention, a skin composition for improving skin wrinkles by inhibiting the activity of collagenase that affects wrinkles by degrading collagen in skin cells by using a fibroblast medium and a functional peptide, and promoting the production of collagen in cells. Can be provided.

1 is a graph comparing the collagenase inhibitory ability according to the cosmetic composition prepared in Examples 1 to 20, Comparative Examples 1 and 2 of the present invention,
Figure 2 is a graph comparing the MMP-1 inhibitory ability according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention and the cosmetic composition prepared in Comparative Examples 2 to 6,
3 is a graph comparing the collagen production ability according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention and the cosmetic composition prepared in Comparative Examples 2 to 6,
Figure 4 is a photograph taken by retardation microscope (LABOMED, Eyecam) of the keratinocytes of the cosmetic composition prepared in Example 11 and Comparative Example 2 of the present invention,
5 is a photograph taken with a digital camera (NIKON D3200, 18-55mm) by observing changes in color and aroma over time of the cosmetic composition prepared in Example 11 and Comparative Example 2 of the present invention,
Figure 6 is a graph measuring the thermal stability according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention,
7 is a graph measuring the light stability according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention,
8 is a graph confirming cytotoxicity according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention by MTT assay in NIH3T3 cells,
9 is a graph confirming the cytotoxicity according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention by MTT assay in HACAT cells.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments. Prior to this, terms or words used in the present specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly explain the concept of terms in order to best explain their own invention. Based on the principle that it can be defined, it should be interpreted as meaning and concept corresponding to the technical idea of the present invention. Therefore, the configuration of the embodiments described herein is only one of the most preferred embodiments of the present invention and does not represent all of the technical idea of the present invention, various equivalents and modifications that can replace them at the time of the present application It should be understood that there may be

The present inventors have made intensive efforts to develop a composition that can effectively prevent or improve skin aging and wrinkles, and as a result, hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2, acetyl tetrapeptide It was confirmed that the composition containing -1 and the conditional broth component of fibroblast as an active ingredient inhibits skin cell regeneration and collagenase activity and promotes intracellular collagen production. The invention was completed.

In addition, the present inventors refer to a patent for a condition medium having excellent skin aging prevention and wrinkle improvement effects (US Pat. No. 8,518,879). In the present invention, the present invention was completed by confirming the effect by developing a composition containing five peptides, which have been widely used because of the condition medium and the traditionally known to improve the skin wrinkles. Therefore, all of the contents disclosed in the US patent application (US Patent No. US 8,518,879) may be a reference for the present invention.

The functional raw materials constituting the cosmetic composition for improving skin wrinkles of the present invention are all raw materials that act on the skin through different mechanisms to help improve skin wrinkles, and therefore, when used in combination, each of the raw materials is used alone. In comparison, the effect can be further enhanced.

Therefore, the present invention is effective in conditioned medium and hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl tetrapeptide-1 from cultured homogeneous foreskin. Disclosed is a cosmetic composition for improving wrinkles comprising as a component.

The living cells secrete proteins and peptides to the outside of cells by including growth factors in the nutrient medium during artificial culture. In the present invention, 'conditioned medium' refers to a medium in which growth factors are secreted through artificial culture of cultured cells. (See US Pat. No. 6,372,494).

The fibroblast may be a cell line designated by ATCC Accession No. PTA-11680 or a cell line designated by PTA-11681.

The fibroblasts can be separated by the following method, but is not limited thereto. For example, fresh tissue samples (eg, human foreskin) are thoroughly washed and chopped under Hank's balanced salt solution (HBSS) to remove serum. The chopped tissue is incubated for 1 to 12 hours in a solution such as trypsin. After such culture, dissociated cells can be suspended and separated via centrifuge.

In the present invention, the condition medium may be a medium cultured by inoculating the fibroblasts in a growth medium.

The growth medium may be any cell culture medium that adequately describes the nutritional needs of the cells to be cultured.

In addition, the growth medium may be supplemented using any component necessary to support a given cell or tissue culture. In addition, serum, such as bovine serum, which is a combination of growth inhibitors, growth promoters, globulins and albumin, may be added if necessary, and the serum should be free of pathogens.

The components of the growth medium are amino acids (both D and / or L-amino acids) such as glutamine, alanine, arginine, asparagine, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine , Tryptophan, tyrosine and valine and derivatives thereof; Acid soluble subgroups such as thiamine, ascorbic acid, ferric compound, ferrous compound, purine, glutathione and monobasic sodium phosphate.

Further components may also include sugars, deoxyribose, ribose, nucleosides, water soluble vitamins, riboflavin, salts, trace metals, lipoacetate salts, phosphate salts, HEPES, phenol red, pyruvate salts and buffers.

In addition, other components often used in media formulations include fat soluble vitamins (including A, D, E and K) steroids and derivatives thereof, cholesterol, fatty acids and lipids Tween 80, 2-mercaptoethanol pyramidine, Serum (fetal, equine, calf, etc.), proteins (insulin, transferrin, growth factor, hormones, etc.), antibiotics (gentamicin, penicillin, streptomycin, amphotericin B, etc.). Various supplements, including nectin, collagen, laminin, tenascin, and the like.

In addition, because many cell constructs themselves produce such growth and adhesion factors and other products in the medium, the medium may be supplemented with growth factors, peptides and other proteins such as adhesion factors.

Other components for the growth medium such as vitamins, growth and adhesion factors, peptides, proteins and the like can be selected by those skilled in the art depending on the particular needs.

Conditioned medium of fibroblasts derived from the cultured homogeneous foreskin of the present invention may be prepared by the following method, but is not limited thereto. For example, 35 to 39 parts by inoculating 0.5 to 5 parts by weight of a medium containing 10 ⁵ to 10 ¹⁰ cfu / ml of fibroblasts from the cell line designated ATCC accession number PTA-11680 or the cell line designated ATCC accession number PTA-11681. It may be carried out by incubating for 1 to 5 days at ℃, preferably inoculated 0.5 to 2 parts by weight with a culture solution of 10 ⁵ to 10 ⁹ cfu / ㎖ may be carried out by incubating for 2 to 4 days at 36 to 38 ℃. In this case, the medium used for the culture may include peptone, sodium acetate hydrate, magnesium sulfate hydrate, manganese sulfate hydrate, Tween 80, ammonium citrate, dipotassium phosphate, and distilled water.

In the present invention, hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl tetrapeptide-1 may be included at 0.005 to 0.1% (w / v), respectively, based on the total composition. The condition medium may be included in 1 to 10% (v / v), preferably hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl tetrapeptide- 1 may be included in 0.005 to 0.05% (w / v), respectively, and most preferably 0.01 to 0.05% (w / v). In addition, the condition medium may preferably be included in 5 to 10% (v / v).

The tripeptide-29 is a small peptide having a molecular weight of 300 or less and consists of the amino acid sequence of Hyp-Pro-Gly. Percutaneous absorption is possible, so it can be efficiently absorbed into the stratum corneum, epidermal layer, hair follicles, etc., so that it is absorbed into the skin to produce collagen, which is known to be very effective in increasing skin elasticity and wrinkle improvement.

The acetyl tetrapeptide-7 is a peptide made by introducing a palmitoyl group (Pal-Gly-Gln-Pro-Arg-OH molecular weight 694.91) into a peptide consisting of four amino acids (Gly-Glnp-Pro-Arg). It is a cosmetic peptide that is effective to increase skin elasticity by increasing skin elasticity, skin regeneration and collagen by increasing palmityl group to increase skin absorption of existing tetrapeptide.

The acetyl peptide peptide-1 and acetyl tetrapeptide-2 are two peptides derived from Thymopoietin (Lamina-associated polypeptide 2; LAP2). Thymopoietin has a nickname of youth hormone. Acetylpeptide peptide-1 and acetyltetrapeptide-2 are peptides made by reconstituting only the essential sequences of thymopoietin. They are involved in the differentiation and growth of epidermal cells of the skin and are effective in preventing skin regeneration and wrinkles. Has been utilized as a cosmetic composition.

In addition, the peptide used as an active ingredient in the composition of the present invention is composed of 2 to 6 amino acid residues very low molecular weight, the condition medium also has a low molecular weight due to fermentation by microorganisms, so the skin penetration rate is very excellent. Therefore, when the composition of the present invention is applied to the skin topically, it is possible to achieve an excellent skin wrinkle improvement effect due to high skin penetration rate.

The cosmetic composition of the present invention can promote the growth of skin fibroblasts, can inhibit the activity of MMP-1, can promote intracellular collagen production.

In addition, the cosmetic composition was confirmed that the effect of inhibiting the activity of collagenase, collagenase, such as collagenase, MMP-1 is very excellent, and also has the effect of promoting the growth of intracellular fibroblasts and collagen production It was confirmed to be excellent.

In addition, it was confirmed that the cosmetic composition is not toxic to human-derived cells, as a result of treating the composition with HaCat cells and NIH3T3 cells at a concentration of 100ng / ml-1000μg / ml, cytotoxicity was not measured and visual Even when the cell morphology was confirmed, no significant change was observed.

In addition, the cosmetic composition exhibits excellent thermal stability even at a temperature of 40 ~ 60 ℃, it was confirmed that it is stable even in daylight conditions. That is, the cosmetic composition means that it can be advantageously applied to products that require long-term storage, such as pharmaceuticals, quasi-drugs and cosmetics.

In addition, as a result of comparing the wrinkle improvement effect of the cream containing the composition of the present patent and the cream of the present invention by requesting a certified clinical institution to confirm the clinical efficacy of the cosmetic composition, in the cream containing the composition according to the present invention It was confirmed that the eye wrinkle improvement effect is significantly better.

Therefore, the present invention can provide a cosmetic composition excellent in inhibiting collagenase, promoting collagen production, improving skin elasticity, and moisturizing effect.

In addition, the cosmetic composition may be a flexible cosmetics, nourishing cosmetics, eye cream, nutrition essence, nutrition cream, massage cream, body cream, cleansing cream, cleansing foam, cleansing water, powder or pack formulation, but is not limited thereto.

In addition, the cosmetic composition according to the present invention may be applied to cosmetic compositions such as gel type, skin type, cream type, ointment type, but is not limited thereto. The composition may be appropriately prepared by known methods by adding appropriate conventional softeners, emulsifiers, thickeners or other materials known in the art, depending on the type thereof.

The gel-type composition may be prepared by adding a softener such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, isotropic alcohol, refined liquor and the like.

The skin type composition may be selected from fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, butylene glycol, glycerin, allantoin, methyl paraben, ideti- 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenate castol oil, poly sorbate 60, purified water and the like can be added.

The cream type composition may be a fatty alcohol such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, lecithin, phosphatidylcholine, phosphatidylethanolamine, sorbatizylserine, sorbatidyl Lipids such as inositol, derivatives thereof, emulsifiers such as glyceryl stearate, sorbitan palmitate, and sobitan stearate, natural fats or oils such as avocado oil, apricot oil, babassu oil, free acid oil, camellia oil, It can manufacture by adding solvent, such as propylene glycol, and purified water.

The ointment type composition may be prepared by adding a softener, an emulsifier and waxes such as microcrystalline lead, paraffin, ceresin, beeswax, braze, petrolatum and the like.

The cosmetic composition of the present invention may further contain at least one skin wrinkle improvement component exhibiting the same or similar function in addition to the active ingredient. The skin wrinkle improvement component may be any one or more selected from the group consisting of vitamin C, retinoic acid, TGF, protein from animal placenta, betulinic acid and chlorella extract, but is not limited thereto. In addition, the cosmetic composition of the present invention may further contain an excipient including a fluorescent substance, a fungicide, a caustic agent, a moisturizer, a fragrance, a fragrance carrier, a protein, a solubilizer, a sugar derivative, a sunscreen agent, a vitamin, a plant extract, and the like. Can be.

Hereinafter, specific examples according to the present invention will be described.

Production Example  Of fibroblasts Conditional  Produce

I. Culture

For the method of securing the fibroblasts (ATCC Accession No. PTA-11680 and PTA-11681) necessary for the preparation of the conditioned medium, refer to the prior patent (US Pat. No. 8,518,879), and then culturing the obtained fibroblasts. Cell suspensions were made. Specifically, a wave bioreactor having a Nunc microhex microcarrier (Nalge Nunc International, Denmark) was used. Nunk microhex is a 2D, flat hexagonal polystyrene carrier having a side length of 125 microns.

II. inoculation

Cellbag (Wave Biotech) was inflated with air and 10% CO ₂ until hard. Medium was added and the inlet and outlet of the filter were clamped. The cell bag was then shaken at an angle of about 7 ° about 15 times per minute, allowing the temperature and pH to equilibrate. The initial volume was about 50% of the final culture volume. Thereafter, the microcarrier and the cell suspension were added. In general, an initial cell density of about 0.1-0.5 × 10 ⁶ cells per ml was added.

The inlet and outlet of the filter were held in a clamped state and the shaking was continued at a speed of about 20 rpm and an angle of about 7 °. The attachment process lasted over several hours or overnight.

III. work

Once the cells were attached, the remaining amount of cell suspension was added to bring the culture to final volume. Cell density, viability and metabolism were monitored during cell growth. Oxygen levels were monitored and rpm and angle were adjusted to the oxygen demand of the culture in the reaction. It is desirable to maintain low rpms and angles while maintaining sufficient oxygen and keeping the microcarriers and cells suspended. Stop shaking and perform medium change. The platform was tilted forward to allow microcarriers and cell complexes to settle within minutes at the bottom edge of the cell bag. The medium was then pumped without removing any microcarriers and up to 90% of the culture volume was removed in this manner. Fresh pre-warmed medium was added and shaking of the cell bag resumed at the previous setting.

IV. Rocking speed

The shake rate was dependent on culture volume, cell density and cellback size cells or culture flasks or Petri dishes. For cell bags 2 L and 10 L, the speed was initially set at about 15-20 rpm. The speed was increased to about 20-25 rpm as the cell density increased.

V. Shake Angle

For cell bags 2 L and 10 L, an initial angle of 6 ° was set. Once the maximum cell density was achieved, it was set at an angle of about 7-8 °.

VI. Ventilation  Aeration rate

The cell bag was kept inflated firmly. A flow rate of less than about 0.5 liters per minute (1 pm) was used while the cell bag was inflated. When cell growth was observed, the flow rate was set to about 0.11 pm for 2 l bags and about 0.21 pm for 10 l cell bags.

VII. Working temperature

Typical operating temperatures for mammalian cells are 36 to 37 ° C.

VIII. pH adjustment

Due to the high gas transport capacity of the wave type bioreactor, the pH can be shifted quickly. The pH was adjusted according to the following procedure.

a. The cell bag was initially inflated with 10% CO ₂ . After inflation, the medium and microcarrier were added to the bioreactor and the inlet and outlet of the air filter were closed. For pH and temperature to fully equilibrate, the cell bag was allowed to shake at about 15 rpm for 1-2 hours. Before inoculation, a sample was taken if necessary to check and adjust the pH.

b. The microcarriers were seeded with cells, where the inlet and outlet of the filter were closed.

c. pH, glucose concentration and cell density are monitored. Once the pH and glucose levels began to drop, the continuous air flow through the headspace was replaced with 5% CO ₂ . When a rapidly growing cell is found, the pH is adjusted to the CO ₂ concentration in the sweep gas.

d. The shaking speed and angle were increased to maintain oxygen concentration.

e. Care was taken when changing the medium used, and the pH was monitored and the CO ₂ concentration was adjusted as the cells adapted to the fresh medium.

IX. Scale up

Scale up according to the following procedure.

a. 500 ml medium was used to fill 2 L cell bags. 13 g microhex carrier was added and pH was equilibrated. Sufficient cell inoculum was added to produce a starting cell count of at least 0.3 × 10 ⁶ cells / ml (total 1.5 × 10 ⁸ cells). The shaking speed was set at about 15 rpm and the shaking angle at about 6 ° for 12 to 24 hours. The system maintained the operating temperature.

b. The next day, 500 ml of medium was added, the rpm was adjusted to about 18 and the angle was adjusted to about 6.

c. Incubation was continued until pH began to drop. The inlet and outlet of the filter were clamped and CO ₂ was added. Oxygen levels in the cultures were monitored and the rpm was adjusted to about 20.

d. Glucose levels and low pH continued for several more days until the medium was used. 50% of the medium was changed and pH was carefully monitored. The rpm was adjusted to about 22 and the angle was adjusted to about 7 °.

e. 50% medium exchange lasted every other day.

Through the same process as described above to prepare a condition medium.

Example  One

Conditioned medium 5v / v% by adding water to the conditioned medium, hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl tetrapeptide-1 of the animal cells prepared in Preparation Example , Hexapeptide-9 0.06w / v%, acetyl tetrapeptide-7 0.01w / v%, tripeptide-29 0.01w / v%, acetyl tetrapeptide-2 0.01w / v% and acetyl tetrapeptide-1 0.01w / v% was prepared. After mixing water to prepare a cosmetic composition.

Example  2

A cosmetic composition was prepared in the same manner as in Example 1, except that hexapeptide-9 0.05w / v% and acetyl tetrapeptide-7 0.02w / v% were mixed in Example 1.

Example  3

A cosmetic composition was prepared in the same manner as in Example 1, except that hexapeptide-9 0.05w / v% and tripeptide-29 0.02w / v% were mixed in Example 1.

Example  4

Cosmetic composition in the same manner as in Example 1 except for mixing hexapeptide-9 0.04w / v%, acetyl tetrapeptide-7 0.02w / v% and tripeptide-29 0.02w / v% in Example 1 Was prepared.

Example  5

Cosmetic composition in the same manner as in Example 1 except for mixing hexapeptide-9 0.03w / v%, acetyl tetrapeptide-7 0.03w / v% and tripeptide-29 0.02w / v% in Example 1 Was prepared.

Example  6

Cosmetic composition in the same manner as in Example 1 except for mixing hexapeptide-9 0.03w / v%, acetyl tetrapeptide-7 0.02w / v% and tripeptide-29 0.02w / v% in Example 1 Was prepared.

Example  7

Cosmetic composition in the same manner as in Example 1 except for mixing hexapeptide-9 0.02w / v%, acetyl tetrapeptide-7 0.03w / v% and tripeptide-29 0.03w / v% in Example 1 Was prepared.

Example  8

A cosmetic composition was prepared in the same manner as in Example 1, except that 0.02w / v% of hexapeptide-9 and 0.04w / v% of acetyl tetrapeptide-7 were mixed in Example 1.

Example  9

A cosmetic composition was prepared in the same manner as in Example 1, except that 0.02w / v% of hexapeptide-9 and 0.04w / v% of tripeptide-29 were mixed in Example 1.

Example  10

A cosmetic composition was prepared in the same manner as in Example 1, except that 0.02w / v% of hexapeptide-9 and 0.05w / v% of acetyl tetrapeptide-7 were mixed in Example 1.

Example  11

Hexapeptide-9 0.02w / v%, acetyl tetrapeptide-7 0.02w / v%, tripeptide-29 0.05w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Example  12

Hexapeptide-9 0.02w / v%, acetyl tetrapeptide-7 0.05w / v%, tripeptide-29 0.02w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Example  13

Hexapeptide-9 0.02w / v%, acetyl tetrapeptide-7 0.03w / v%, tripeptide-29 0.04w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Example  14

Hexapeptide-9 0.02w / v%, acetyl tetrapeptide-7 0.04w / v%, tripeptide-29 0.03w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Example  15

Hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.07w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide-1 0.005w / v% in Example 1 were mixed A cosmetic composition was prepared in the same manner as in Example 1, except that.

Example  16

In Example 1, tripeptide-1 hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.06w / v%, tripeptide-29 0.02w / v%, acetyl tetrapeptide-2 0.005w / v% And a cosmetic composition was prepared in the same manner as in Example 1 except for mixing 0.005w / v% of acetyl tetrapeptide-1.

Example  17

Hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.05w / v%, tripeptide-29 0.03w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Example  18

Hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.04w / v%, tripeptide-29 0.04w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Example  19

Hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.03w / v%, tripeptide-29 0.02w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Example  20

Hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.02w / v%, tripeptide-29 0.06w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl tetrapeptide in Example 1 A cosmetic composition was prepared in the same manner as in Example 1, except that -1 0.005w / v% was mixed.

Comparative example  One

Commercial epigallocatechin gallate (EGCG) was dissolved in water to prepare a cosmetic composition at 0.05w / v%.

Comparative example  2

5v / v% of the cosmetic composition was prepared by adding water to the conditioned medium prepared in Preparation Example.

Comparative example  3

Hexapeptide-9 0.02w / v%, acetyl tetrapeptide-7 0.02w / v%, tripeptide-29 0.05w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl A cosmetic composition was prepared by mixing tetrapeptide-1 0.005w / v%.

Comparative example  4

Hexapeptide-9 0.02w / v%, acetyl tetrapeptide-7 0.03w / v%, tripeptide-29 0.04w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl A cosmetic composition was prepared by mixing tetrapeptide-1 0.005w / v%.

Comparative example  5

Hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.04w / v%, tripeptide-29 0.04w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl A cosmetic composition was prepared by mixing tetrapeptide-1 0.005w / v%.

Comparative example  6

Hexapeptide-9 0.01w / v%, acetyl tetrapeptide-7 0.02w / v%, tripeptide-29 0.06w / v%, acetyl tetrapeptide-2 0.005w / v% and acetyl A cosmetic composition was prepared by mixing tetrapeptide-1 0.005w / v%.

Table 1 below is a table showing the ratio of each composition of Examples 1 to 20 and Comparative Examples 2 to 6.

Experimental Example  1: of composition Collagenase  Confirmation of activity inhibitory effect

Experiments on the ability to inhibit in vitro collagenase activity in the following manner to confirm the skin wrinkle improvement effect of the wrinkle improving cosmetic composition prepared in Examples 1 to 20, Comparative Examples 1 and 2 Was carried out, and the results are shown in FIG.

Experimental Method

1. Wrinkle improvement cosmetic composition prepared in Examples 1 to 20 and Comparative Examples 1 to 6 was prepared by dissolving in phosphate buffer (100, 50, 25 ppm)

2. Prepare 300 μl / 48 wells of the test sample by diluting at 48well to the appropriate concentration range using solvent (DMSO).

3. Prepare a 15 ml glass tube and dispense the solution in the order shown in Table 2 below.

4. Terminate the reaction by adding 400 μl / tube of 25 mM citric acid.

5. Add 1.5 ml / tube of ethyl acetate, vortex and leave for 10 minutes.

6. Take the supernatant, transfer to 150 mg / ep-tube of sodium sulfate, and centrifuge (10,000 rpm, 3 minutes) after vortexing.

7. Take 300 μl / ep-tube of supernatant, transfer to quartz 96well plate, and measure absorbance at 320 nm.

1 is a graph comparing the collagenase inhibitory ability according to the cosmetic composition prepared in Examples 1 to 20, Comparative Examples 1 and 2 of the present invention.

Referring to Figure 1, the result of the in vitro collagenase activity inhibition test was a higher cola compared to the cosmetic composition (Comparative Example 2) prepared only a control medium of the cosmetic composition (Examples 1 to 20) mixed with a medium and a peptide It was confirmed that it exhibits a genease inhibitory activity, and there is no collagenase inhibitory activity of the conditioned medium itself. In particular, it was confirmed that Examples 11, 13, 18 and 20 are excellent in the inhibition rate of collagenase activity. In addition, the collagenase inhibition rate of the cosmetic composition (Comparative Example 1) prepared using EGCG confirmed the inhibition rate similar to the cosmetic composition (Examples 1 to 20) in which the medium and the peptide were mixed.

Experimental Example  2 : MMP -1 (Matrix Metalloproteinase-1) Activity Inhibition Experiment

Substances that inhibit the production of matrix methaloprotease-1 (MMP-1, collagenase) measure the inhibitory activity of MMP-1 production on raw materials because it protects collagen and maintains the mechanical properties of skin tissue to prevent elasticity and sagging. It can be evaluated that the active material can be useful for improving wrinkles and developing cosmetics for elastic skin. Examples (Examples 11, 13, 18 and 20), and the MMP-1 activity inhibition test for the cosmetic composition of Comparative Examples 2 to 6 was carried out according to the following method and the results are shown in FIG. Retinol was used as a control.

Experimental Method

Human dermal fibroblst (HDF) cells were cultured with DMEM (Dulbecco's modified Eagle's media, Sigma) added with 10% fetal bovine serum (Sigma) at 37 ° C and 5% CO _2. Incubated at.

(2) After incubating the cells at a concentration of 2 X 10 ⁵ cells / well in a 24-well plate and confirming the cell attachment, the control group was treated with nothing but only the solvent, and the dish was prepared with 9 experimental groups and the control group with retinol. Was treated to a concentration of 10 ug / ml.

(3) After the test substance was added to each dish, the plate was incubated for 2 days. 2 days later, the medium was collected through M atrix Metalloproteinase-1 (MMP- 1), Human, biotrak, ELISA kit (GE healthcare RPN2610) was measured to produce inhibition of MMP-1.

Figure 2 is a graph comparing the MMP-1 inhibitory ability according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention and the cosmetic composition prepared in Comparative Examples 2 to 6.

Referring to Figure 2, it was confirmed that the amount of expression of MMP-1 produced in the cell is reduced compared to the control retinol in the cosmetic composition (Examples 11, 13, 18 and 20) made of a condition medium and peptide. It has been found that there is a difference in efficacy depending on the combination and composition of ingredients. The efficacy of Example 11 was the best and the cosmetic composition prepared using the peptide and the conditioned medium, the cosmetic composition prepared using only the conditioned medium (comparative 2) and the cosmetic composition prepared using only the peptide (Comparative Examples 3 to 6) ) Showed higher MMP-1 expression inhibition.

From these results, when UV, which causes MMP-1 production, is applied as a factor, it is more useful to mix various combinations than when using a single raw material, which inhibits MMP-1 production. It is thought to be able to suppress wrinkles.

Experimental Example 3: cell  My collagen Generating ability  evaluation

Intracellular collagen production test was carried out for the Examples (Examples 11, 13, 18 and 20) and Comparative Examples 2 to 6 according to the following method, the results are shown in FIG. TGF-b was used as a control.

Experimental Method

(1) Human dermal fibroblst (HDF) cells to be used for the test were obtained by adding 1 × LSGS (Low Serum Growth Supplement, cascade biologics S-003-10) to Medium 106 (cascade biologics, M-106-500). The medium was incubated at 37 ° C. and 5% CO ₂ .

(2) After incubating the cells at a concentration of 1 × 10 ⁵ cells / well in a 24-well plate and confirming the cell attachment, the control group was treated with nothing but only the solvent, and the dish was prepared with 9 experimental groups and a control group with TGF. The treatment was carried out to a concentration of 10 μg / ml.

(3) After the test substance was added to each dish, the plate was incubated for 2 days. After 2 days, the culture medium was collected and the amount of collagen production was measured using Procollagen Type I C-peptide (hereinafter PIP) EIA kit (takara MK101).

Figure 3 is a graph comparing the collagen production ability according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention and the cosmetic composition prepared in Comparative Examples 2 to 6.

3, in the case of the cosmetic composition (Examples 11, 13, 18, and 20) prepared by mixing the medium and the peptide, it was confirmed that the amount of collagen was increased in comparison with TGF, which is a control group. It was confirmed that there is a difference depending on the combination and composition of the raw material components. In addition, in the case of the cosmetic composition (Examples 11, 13, 18 and 20) prepared by mixing the medium and the peptide, it was confirmed that the amount of collagen production was increased compared to the cosmetic composition (Comparative Examples 3 to 6) prepared by mixing only the peptide. Can be.

From these results, it is thought that using a combination of various combinations of collagen production in the skin is more useful than in the case of using a single raw material, thereby increasing collagen production and suppressing wrinkles of the skin.

Experimental Example  4: keratinocytes Regenerative  evaluation

 Various intercellular and intracellular metabolisoms act on the regeneration of damaged skin cells due to various causes. Through the wound healing assay, which is an experiment that can activate the metabolism to promote the growth of damaged skin cells and improve the cell recovery ability, the cell regeneration induction effect of Example 11 and Comparative Example 2 was confirmed according to the following method. 4 is shown.

Experimental Method

(1) Cut the middle of the human skin keratinocyte line grown to 100% fill into a 6 well plate using a tip and then wash twice with PBS.

(2) The cosmetic composition prepared in Example 11 and Comparative Example 2 was treated to 10, 5, 2.5, 1.3% concentration in the whole cell culture medium and the experiment time was based on a total of 12, 24 hours, each time The extent of the wound recovery during the imaging was confirmed.

Figure 4 is a photograph of the regeneration ability of keratinocytes of the cosmetic composition prepared in Example 11 and Comparative Example 2 of the present invention with a phase contrast microscope (LABOMED, Eyecam).

Functional peptide N in human skin keratinocytes (HaCaT) also showed the same effect as the positive control vitamin C, and showed the same or more skin regeneration induction effect than commercial products (not shown).

Referring to Figure 4, as a result of confirming the keratinocyte regeneration ability was confirmed that the potency of the keratinocyte regeneration ability is different depending on the concentration of the treated mixture and there is a difference in the composition and composition of the functional ingredient in the composition within the experimental group. It became. In particular, it can be seen that the cosmetic composition (Example 11) prepared by mixing the conditioned medium and the peptide was superior to the cellular regeneration ability compared to the cosmetic composition (Comparative Example 2) prepared using only the conditioned medium. When mixed, it is judged that there is a synergistic effect on cell regeneration. Therefore, from the above results, the production of keratinocytes of skin may be more useful for the regeneration of wounded skin by using a combination of various combinations than in the case of using a single raw material. .

Experimental Example 5: skin  Experiment to confirm stability of wrinkle improving composition

In order to confirm the thermal and light stability of the wrinkle improving composition of the present invention, the cosmetic compositions prepared in Examples 11, 13, 18, 20 and Comparative Example 2 were experimented according to the following method, and the results for each 5 to 7 are shown.

Experimental Method

The composition was dissolved in 50mM Tris-HCl (pH 8.0) buffer solution so that the wrinkle improving composition was 10 mg / mL, aliquoted into glass vials, and stored at 50 ° C for 7, 14, 21, 28, 35, 42 and 60 days. Loss of the peptide component in the composition by heat was measured and stability was determined in daylight conditions by the same method.

5 is a photograph taken with a digital camera (NIKON D3200, 18-55mm) by observing changes in color and aroma over time of the cosmetic composition prepared in Example 11 and Comparative Example 2 of the present invention, Figure 6 The thermal stability according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention is a graph, Figure 7 is a light according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention It is a graph measuring stability.

Referring to FIG. 5, there was no change in color and odor until 60 days, and referring to FIGS. 6 to 7, it showed that peptide loss of less than 10% by 60 days had high stability in thermal and light stability. have.

Experimental Example  6: cytotoxicity test

In order to confirm whether the composition of the present invention is toxic to keratinocytes, the experiments were conducted in the following manner with respect to the cosmetic composition prepared in Examples 11, 13, 18 and 20, and the results are shown. 8 and 9, respectively.

Experimental Method

Cytotoxicity was measured using the MTT assay. HACAT cells and 3T3 cells were seeded in a 24 well plate so as to have a cell number of 4 X 10 ⁴ cells / 24 well per well and incubated at 37 ° C. and 5% CO ₂ for 24 h. After washing twice with PBS and incubated with a synthetic tranexamic acid dipeptide concentration (1mg ~ 1ug / mL) was incubated for 24 h. After incubation, MTT 0.5% in DPBS was mixed with the culture medium at 1: 9 (v / v), and then incubated in a CO ₂ incubator for 2 hours, and the resulting formazan was dissolved in DMSO and measured at 570 nm using ELISA. .

8 is a graph confirming the cytotoxicity according to the cosmetic composition prepared in Examples 11, 13, 18 and 20 of the present invention by MTT assay in NIH3T3 cells, Figure 9 is Examples 11, 13, 18 and Cytotoxicity according to the cosmetic composition prepared in 20 is a graph confirmed by MTT assay in HACAT cells.

Referring to FIGS. 8 and 9, for the HaCat cells, which are a kind of keratinocyte lines, and the NIH3T3 cells, which are a type of fibroblasts, a decrease in the number of cells due to the composition treated from low to high concentrations was not observed, and microscopic observation of the cells was observed. No change was observed at. Through this, it can be seen that the wrinkle improving composition of the present invention is not toxic to skin-related cells at all, thereby predicting that the wrinkle improving composition will not have a great effect even when treated on the skin.

  Preferred embodiments of the present invention described above are disclosed to solve technical problems, and those skilled in the art to which the present invention pertains will be capable of various modifications, changes, additions, etc. within the spirit and scope of the present invention. Such changes, modifications and the like should be considered to be within the scope of the following claims.

Claims (7)

Conditioned medium of fibroblasts derived from cultured homogeneous foreskin and hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl tetrapeptide-1 as active ingredients Cosmetic composition for improving wrinkles. The method of claim 1,
The fibroblast is a cosmetic line characterized in that the cell line designated by ATCC accession number PTA-11680 or a cell line designated by PTA-11681. The method of claim 1,
Hexapeptide-9, acetyl tetrapeptide-7, tripeptide-29, acetyl tetrapeptide-2 and acetyl tetrapeptide-1 for the total composition were included in 0.005 to 0.1% (w / v), respectively.
The conditional medium is a cosmetic composition, characterized in that contained in 1 to 10% (v / v). The method of claim 1,
The cosmetic composition is a cosmetic composition, characterized in that to promote the growth of skin fibroblasts. The method of claim 1,
The cosmetic composition is a cosmetic composition, characterized in that to inhibit the activity of MMP-1. The method of claim 1,
The cosmetic composition is a cosmetic composition, characterized in that to promote the production of collagen in the cell. The method of claim 1,
The cosmetic composition is a cosmetic composition characterized in that it has a flexible cosmetics, nourishing cosmetics, eye cream, nutrition essence, nutrition cream, massage cream, body cream, cleansing cream, cleansing foam, cleansing water, powder or pack formulation.

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