CN110547996A

CN110547996A - Preparation containing horse placenta extract as effective component and skin care cosmetic containing the same

Info

Publication number: CN110547996A
Application number: CN201910152418.7A
Authority: CN
Inventors: 若命浩二; 圆田照夫
Original assignee: Cosmetics Research Institute Co Ltd
Current assignee: Cosmetics Research Institute Co Ltd
Priority date: 2018-05-30
Filing date: 2019-02-28
Publication date: 2019-12-10
Also published as: JP6739472B2; JP2019206495A

Abstract

The invention provides a preparation which is mild and safe to human skin, activates mitochondria, promotes the generation of type I collagen, and has excellent wound healing, and a skin care cosmetic using the same. A preparation containing horse placenta hydrolysate as effective component and skin care cosmetic containing the same are provided, wherein the horse placenta hydrolysate is preferably horse placenta hydrolysate hydrolyzed with fruit derived cysteine protease, and the horse placenta hydrolysate preferably contains peptide as the most.

Description

Preparation containing horse placenta extract as effective component and skin care cosmetic containing the same

Technical Field

The present invention relates to a preparation containing horse placenta hydrolysate as an active ingredient and a skin care cosmetic using the same, and more particularly, to a preparation containing horse placenta hydrolysate as an active ingredient, which contains 1 or 2 or more kinds of substances selected from the group consisting of a mitochondrial activator, a type I collagen production promoter and a wound healing agent, and a skin care cosmetic using the same.

Background

The placenta (placenta) is an organ that is formed in the uterus and connects a mother and a fetus when a female with a placental system or the like is pregnant. In recent years, skin external preparations containing extracts derived from the placenta of livestock have been developed (patent documents 1 and 2).

Patent document 1 discloses the following: the placenta extract improves wound healing effect of saikosaponin b1 and b2, and is obtained from placenta of mammal except human. Patent document 2 discloses the following: the placenta extract is an extract obtained from the placenta of a non-human mammal, particularly a bovine placenta, and has a wound healing promoting effect, and generally contains amino acids, peptides, nucleic acid bases, inorganic substances, vitamins, and the like as components.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. 5-262635

Patent document 2: japanese unexamined patent publication Hei 10-101545

Disclosure of Invention

Technical problem to be solved by the invention

However, the placenta extracts disclosed in patent documents 1 and 2 are extracts of placenta and are not hydrolysates. That is, the wound healing effects of the placental extracts disclosed in patent documents 1 and 2 are currently brought about by growth factors such as EGF (epithelial cell growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor) and the like known to be contained in the placental extract (placental extract), and in hydrolysates, since these growth factors are proteins, they are decomposed and inactivated, and thus are different from the wound healing effects brought about by hydrolysates. Further, patent documents 1 and 2 do not disclose that horse placenta hydrolysate activates mitochondria and promotes the production of type I collagen.

The purpose of the present invention is to provide a preparation that is mild and safe to human skin, and that is excellent in mitochondrial activation, promotion of type I collagen production, and wound healing, and a skin care cosmetic using the same.

Means for solving the problems

The inventors of the present invention conducted intensive studies and found that: horse placenta hydrolysate, particularly horse placenta hydrolysate hydrolyzed by cysteine protease derived from fruits, is excellent in mitochondrial activation, promotion of type I collagen production, and wound healing, and the respective inventions described below have been completed.

(1) a preparation containing as an active ingredient 1 or 2 or more horse placenta hydrolysates selected from the following (a), (b) and (c),

(a) A mitochondrial activator,

(b) I type collagen production promoter,

(c) A wound healing agent.

(2) A preparation contains horse placenta hydrolysate as effective component, and has effects in increasing mitochondria amount in human cell in short term and enhancing mitochondria activity in long term.

(3) A preparation contains horse placenta hydrolysate as effective component, and is used for promoting cell proliferation and improving migration activity of human body to cure wound.

(4) The preparation according to any one of (1) to (3), which is a horse placenta hydrolysate hydrolyzed by a cysteine protease derived from a fruit.

(5) The preparation of any one of (1) to (4), wherein the horse placenta hydrolysate contains a peptide as a component in the largest amount.

(6) the preparation of (5), wherein the horse placenta hydrolysate contains, as a constituent component, at most a peptide having a molecular weight of 1000 or more and less than 3000.

ADVANTAGEOUS EFFECTS OF INVENTION

The preparation of the present invention is mild and safe to human skin, and is excellent in mitochondrial activation, promotion of type I collagen production, and wound healing, and a skin care cosmetic using the same.

Drawings

FIG. 1 is a chromatogram showing the molecular weight distribution of equine placenta hydrolysate measured by High Performance Liquid Chromatography (HPLC).

FIG. 2 is a graph showing the number of cells when normal human fibroblasts (human cells) were cultured in a medium to which a horse placenta hydrolysate, a low molecular weight high-grade gelatin derived from a pig, and a medium molecular weight high-grade gelatin derived from a pig were added.

FIG. 3 is a graph showing the number of mitochondria in normal human fibroblasts (human cells) cultured in a medium to which a horse placenta hydrolysate, a low molecular weight high-grade gelatin derived from a pig, and a medium molecular weight high-grade gelatin derived from a pig are added.

FIG. 4 is a graph showing the activity of mitochondria in normal human fibroblasts (human cells) cultured in a medium to which a horse placenta hydrolysate, a low molecular weight high-grade gelatin derived from a pig, and a medium molecular weight high-grade gelatin derived from a pig are added.

FIG. 5 is a graph showing the amount of type I collagen in normal human fibroblasts (human cells) cultured in a medium to which a horse placenta hydrolysate, a low molecular weight high-grade gelatin derived from a pig, and a medium molecular weight high-grade gelatin derived from a pig are added.

FIG. 6 is a graph showing the results of cell migration tests in the case where horse placenta hydrolysate was not added and in the case where horse placenta hydrolysate was added (100. mu.g/mL).

FIG. 7 is a view showing a culture process in a growth example of a human epidermal model.

FIG. 8 is a graph (a) showing a comparison between a cross section of a human epidermal MODEL (LabCyte EPI-MODEL) and a cross section of a human normal skin (eyelid) and a graph (b) showing a configuration and a state of subculture in the three-dimensional culture kit.

Fig. 9 is a graph showing the results of the skin growth ability confirmation test. In the figure, (a) shows a control, (b) shows a case where a horse placenta hydrolysate of 5% (w/w) was added, and (c) shows a case where a horse placenta hydrolysate of 50% (w/w) was added.

Fig. 10 is a graph showing the results of the skin irritation test. In the figure, (a) shows the results when 5% SLS was used as a sample; (b) shows the results obtained when 50% (w/w) of horse placenta hydrolysate was used as a sample; (c) the results are shown when 5% (w/w) of horse placenta hydrolysate was used as a sample.

Detailed Description

The preparation of the present invention will be described in detail below. The preparation of the present invention contains horse placenta hydrolysate as an active ingredient, and is 1 or 2 or more kinds selected from (a) mitochondrial activators, (b) type I collagen production promoters, and (c) wound healing agents.

The placenta used in the present invention is a horse placenta (placenta derived from horses). Horses are classified as odd-leg horses because they have a body temperature 5 to 6 ℃ higher than pigs or cattle and are not concerned with parasites, and horses are not affected by diseases of even-leg animals such as mad cow disease (bovine spongiform encephalopathy) or foot and mouth disease. Further, horses are not ruminated like cattle, and their internal organs are clean and sensitive, so they cannot tolerate additives or hormones, and are not as versatile as other livestock, and are considered to be hardly allergenic because of their very low antigenicity, which is superior to pig-derived placentas and cow-derived placentas. The horse placenta used in the present invention is not particularly limited as long as it is a horse placenta, and it is preferable because it is easy to use a ground product (a minced product). Further, it is considered that blood or other parts are attached to or mixed with the horse placenta, and therefore, it is preferable to perform washing or the like in advance.

In the present invention, the enzyme used for hydrolysis of horse placenta is not particularly limited as long as it is a protease, but a cysteine protease (cysteine endopeptidase) is preferable, and a cysteine protease derived from fruit is more preferable. Examples of such proteases include: papain (papain), chymopapain (chymopain), asclepiasin (asclepiain), bromelain (broomelain), zingibain (zingibain), ficin (ficin), kiwi protease (actindiain), diarylheptanoid metabolites (curcumin derived from turmeric), and the like.

The preparation of the present invention has an effect of activating mitochondria of cells. That is, the formulations of the present invention are mitochondrial activators. The cell is not particularly limited as long as it is a mammalian cell, and is preferably a human cell, and more preferably a normal human fibroblast. The method for detecting whether or not the mitochondria of the cell are activated is not particularly limited, and a method that can be appropriately selected by a person skilled in the art can be used. In the examples of the present specification, detection was performed by using cell staining with rhodamine and fluorescence intensity value measurement.

In addition, the preparation of the present invention has an effect of promoting the production of type I collagen in cells. Namely, the preparation of the present invention is a type I collagen production promoter. The method for detecting whether or not the production of type I collagen in a cell is promoted is not particularly limited, and a method that can be appropriately selected by a person skilled in the art can be used. In the examples of the present specification, detection is performed by an assay using an enzyme-linked immunosorbent assay (ELISA).

further, the preparation of the present invention has an effect of healing wounds of cellular tissues. That is, the preparation of the present invention is a wound healing agent. As described above, since the placenta extract contains known growth factors such as EGF (epithelial cell growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor) and the like as proteins, and is decomposed and inactivated by hydrolysis reaction, the wound healing agent of the present invention can be said to have a different wound healing effect from that of the existing placenta extract. The method for confirming the wound healing effect in the cells is not particularly limited, and a method that can be appropriately selected by those skilled in the art can be used. In the examples of the present specification, the confirmation was carried out by a cell migration test using the scratch method.

further, as will be understood from the examples described later, the horse placenta hydrolysate of the present invention is a hydrolysate containing a peptide as a maximum component. As will be understood from the examples described later, the horse placenta hydrolysate of the present invention contains, as a constituent component, at most a peptide having a molecular weight of 1000 or more and less than 3000. In the present invention, "the peptide is contained most as a constituent" means that the peptide is contained most as a constituent based on the total weight of the constituent (the peptide is contained most as a weight% of the constituent), and "the peptide is contained most as a constituent" in a substitution manner "the peptide as a constituent (in the horse placenta hydrolysate) is the first composition having the maximum composition ratio".

In addition, the skin care cosmetic of the present invention contains the agent of the present invention, a moisturizing agent and a preservative. That is, the present invention is characterized in that a moisturizer and a preservative are selected and added to a specific preparation to be applied to a skin care cosmetic, thereby providing a skin care cosmetic excellent in storage stability.

The humectant used in the present invention may be selected within a range that does not impair the characteristics of the preparation of the present invention, and as such humectants, for example: polyhydric alcohols such as glycerin, 1, 3-butanediol, 1, 3-propanediol, 1, 2-pentanediol, polyglycerol, and sorbitol, mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, and salts thereof.

The preservative used in the present invention may be selected within a range that does not impair the characteristics of the preparation of the present invention, and examples of such preservatives include: benzoic acid, sodium benzoate, paraben, sorbic acid, potassium sorbate, phenoxyethanol, 1, 2-hexanediol, 1, 2-octanediol, glyceryl caprylate, and ethylhexyl glycerin.

The method for producing the preparation of the present invention comprises the following steps (i) to (iv): (i) a step (hydrolysate preparation step) of adding a hydrolase to a horse placenta and hydrolyzing the mixture to prepare a hydrolysate; (ii) a step (enzyme inactivation step) of inactivating the hydrolase after the preparation of the hydrolysate; (iii) a step of performing isoelectric point treatment (isoelectric point treatment step); and (iv) a step of separating and filtering the supernatant (supernatant separation and filtration step).

In the step (i) of preparing the hydrolysate, the temperature setting or the time required for the hydrolysis reaction may be appropriately set within a range that does not affect the hydrolysis reaction. In addition, the temperature setting or the setting of the time required for inactivating the enzyme in the (ii) enzyme inactivation step may be appropriately set within a range in which the enzyme can be inactivated.

On the other hand, (iii) setting of the substance and pH used in the isoelectric point treatment step, and (iv) selection of the membrane filter used in the supernatant liquid separation filtration step, for example, selection of the pore size, may be appropriately selected within a range in which these steps can be performed.

In the present invention, the skin care cosmetic may contain, as appropriate, at least 1 component selected from pearl gloss materials, antioxidants, pigments, thickeners, and pH adjusters, which are generally used in the cosmetic field, within a range not to impair the characteristics of the present invention. In addition, aromatic, antibacterial, various oily components, ultraviolet absorbent, active oxygen remover, antioxidant, antimicrobial agent, pilatory, mineral, and amino acid can be used. Suavia, a known whitening ingredient may be used in combination.

Examples

The formulation of the present invention is described below based on examples. The technical scope of the present invention is not limited to the embodiments shown in the examples.

1. Preparation of horse placenta hydrolysate

Horse placenta hydrolysate used in this example was prepared. 100 parts by weight of a minced placenta of a pure horse produced in hokkaido, which was preserved in advance under freezing, was subjected to semi-thawing, 300 parts by weight of purified water was added, and after thawing at room temperature, 0.08 part by weight of papain and 0.12 part by weight of bromelain, which are cysteine proteases derived from fruits, were added, and hydrolysis reaction was performed at 45 to 55 ℃ for a total of 6 hours. Subsequently, the protease was inactivated and water-cooled, treated with an isoelectric point treatment using lactic acid (pH4.37) and activated carbon (powder: 0.25 part by weight), and allowed to stand at room temperature overnight. Then, the supernatant was separated, filtered using a membrane filter (pore size: about 3 μm), neutralized with 10% (v/v) sodium hydroxide (pH7.3), and then filtered using a membrane filter (pore size: 1.0 μm and pore size: 0.45 μm), to obtain a horse placenta hydrolysate.

2. General nutritional analysis test of horse placenta hydrolysate

General nutritional analysis tests were performed on the horse placenta hydrolysate prepared in 1. The analytical test was entrusted to Japanese food analysis center, a general treasury judge. Specific test methods are as follows.

[2-1] test method for general Nutrition analysis test

The analytical test items were defined as water, protein, lipid, ash, carbohydrate, energy, sodium, and salt equivalent, and the values were determined by the atmospheric pressure heat drying method for water, the combustion method (nitrogen-protein conversion factor: 6.25) for protein, the acid decomposition method for lipid, the direct ashing method for ash, the formula (100- (water + protein + lipid + ash)) for food (hei 27 years condo 10) for carbohydrate, the energy conversion factor (protein 4: lipid 9: carbohydrate 4) for energy food (hei 27 years condo 10), and the formula (sodium × 2.54) for sodium atomic absorption photometry and salt equivalent. The results of the general nutritional analysis tests on horse placenta hydrolysate are shown in table 1.

[ Table 1]

[2-2] results

As shown in Table 1, the horse placenta hydrolysate of the present example contained 88.1g/100g of protein as a component. From this, it was found that the horse placenta hydrolysate of the present example contains most proteins, i.e., peptides, as constituent components.

3. Analytical test of molecular weight distribution

The horse placenta hydrolysate prepared in 1 was subjected to an analysis test of molecular weight distribution. The analytical test was entrusted to Japanese food analysis center, a general treasury judge. Specific test methods are as follows.

[3-1] preparation of Standard solution

Molecular weight standards (proteins and peptides) shown in table 2 below were dissolved in a mixture (55: 45:01 by volume) of water, acetonitrile and trifluoroacetic acid to prepare a standard solution. Wherein in Table 2, R1 represents a glycine residue, R2 represents a tyrosine residue, and R3 represents an arginine residue.

[ Table 2]

[3-2] preparation of test solution

The horse placenta hydrolysate prepared in the step 1 is collected by 0.02g, and 10mL of a mixture of water, acetonitrile and trifluoroacetic acid (volume ratio is 55:45:01) is added. After standing overnight at room temperature, the mixture was filtered through a membrane filter (pore size: 0.45 μm) to prepare a test solution.

[3-3] analysis by HPLC

For the test solution obtained in [3-2], an analytical test (size exclusion chromatography) of molecular weight distribution was performed by High Performance Liquid Chromatography (HPLC) based on the standard solution obtained in [3-1 ]. The operating conditions of HPLC are shown below, and the elution time of the base molecular weight is shown in Table 3 below.

The machine model is as follows: LC-20AD (Shimadzu corporation)

A detector: ultraviolet visual detector SPD-20A (Shimadzu corporation)

Column: TSKgel G2500PW_XL、(Tosoh corporation)

Column temperature: 40 deg.C

Mobile phase: the above-mentioned mixed solution of water, acetonitrile and trifluoroacetic acid

Flow rate: 0.5 mL/min

measuring wavelength: 220nm

Injection amount: 20 μ L

[ Table 3]

The relationship (calibration curve) between the molecular weight (M) and the elution time (T) is as follows.

logM＝－0.3154103T+7.6928709

[3-4] results

From the above, the chromatogram measured for the test solution is shown in fig. 1, and the molecular weight distribution of the horse placenta hydrolysate as the result of the analysis test is shown in table 4. The molecular weight distribution shown in Table 4 is a percentage showing the peak area of the chromatogram of FIG. 1.

[ Table 4]

Molecular weight range	Results	Lower limit of quantification
			more than 6000	11％	-
3000 or more and less than 6000	17％	-
			1000 or more and less than 3000	33％	-
500 or more and less than 1000	11％	-
			Less than 500	28％	-
Total up to	100％

as shown in fig. 1 and table 4, the peak of the molecular weight distribution of the horse placenta hydrolysate of the present example was 1000 or more and less than 3000. Among them, the molecular weight standards of this example are proteins and peptides. From this, it was found that the horse placenta hydrolysate of the present example contains peptides having a molecular weight of 1000 or more and less than 3000 as the maximum component.

4. Peptide detection Using Dye C57

The concentration of a Peptide consisting of 2 to 20 amino acids in a sample was measured by fluorescence using Dye C57 which changes to a fluorescent substance in the presence of an aminocarboxylic acid and using Peptide Assay Kit PEP200 (ProFoldin). Specific test methods are as follows.

[4-1] peptide assay

1mL of 100. mu.g/mL Phosphate Buffered Saline (PBS) solution of the horse placenta hydrolysate prepared in item 1 or the horse placenta material itself was added dropwise to 3 spots of a 96-well plate, and 1mL was added dropwise to 1 spot of the 96-well plate as a blank. To each of them, 25. mu.L of 1 XDdye C57 solution adjusted to 100. mu.L in PBS concentration was added, mixed and diluted, and then allowed to stand at room temperature for 15 minutes, and then measured by fluorescence under excitation at 475nm and measurement at 500 to 550nm using a microplate reader (Promega Glo Max DISCOVER GM 3000).

[4-2] results

The fluorescence intensity of the horse placenta material itself was 248 (after blank correction), while the fluorescence intensity of the horse placenta hydrolysate of the present example was 626 (after blank correction). From this, it was found that the horse placenta hydrolysate of the present example obtained by hydrolyzing horse placenta with a fruit-derived cysteine protease contained about 8 times as much peptide as the horse placenta material itself.

5. Verification test for activation of mitochondria and promotion of type I collagen production

The activity (amount-activity) of mitochondria by horse placenta hydrolysate, low molecular weight high-grade gelatin derived from pig (average molecular weight about 8,000) and medium molecular weight high-grade gelatin derived from pig (average molecular weight about 60,000) for medical use was verified using normal human fibroblasts (human cells), and the promotion of type I collagen production was verified. The analytical test was entrusted to ACEL corporation. Specific test methods are as follows. Among these, the reason why low molecular weight high-grade gelatin derived from pig and medium molecular weight high-grade gelatin (collagen) derived from pig were used as comparative samples with horse placenta hydrolysate was as follows: (1) other company-made samples, which are hydrolytic species derived from the placenta of other horses or other mammals, clearly showing the molecular weight of the peptide, are not present; (2) collagen is generally widely distributed in the living body as a constituent of mammalian proteins, and is also assumed to be present in the placenta; (3) the low molecular weight and high-grade gelatin derived from swine and the medium molecular weight and high-grade gelatin (collagen) derived from swine are subjected to quality control in a medical grade, and are clearly displayed as low and medium molecular weights, and thus can be judged as comparative objects.

[5-1] culture conditions for Normal human fibroblasts (human cells)

After recovery of normal human fibroblasts (human cells), they were cultured in minimal medium (DMEM (nacalai tesqu, Cat. No.08456-65), 10% FBS (biorest, Cat. No. S1820, Lot No.516536) and 1% antibiotics) in CO₂Incubator (5% CO)₂37 ℃ C.) until the desired number of cells is reached. After the culture, the cells were detached using trypsin/EDTA, and the number of the cells was measured and used for the test. Wherein the composition of the test medium is DMEM, 1% FBS and 1% antibiotics.

[5-2] Regulation of test substance

The horse placenta hydrolysate prepared in the above item 1, low molecular weight high-grade gelatin derived from pig, and medium molecular weight high-grade gelatin derived from pig were added to the test medium at a concentration of 25mg/mL, and after filtration sterilization, the mixture was used as a concentrated sample solution. As a positive control, resveratrol (SIGMA company; Cat. No. R5010-100MG) was dissolved in DMSO so as to be 200mM, and the solution was used as a concentrated solution after filtration sterilization. Similarly, vitamin C (ascorbic acid, Wako pure chemical industries, Ltd.; 013-.

[5-3] cell culture

The adjusted normal human fibroblasts (human cells) were seeded at 1X 10 in the minimal medium⁴Cells were cultured in 0.1 mL/well/96-well plate (Greiner Bio-one company; Black plate for fluorescence observation of Cat. No. 655090), and after 24 hours, the culture supernatant was replaced with a minimal medium containing the test substance, and the cells were cultured. The culture was carried out for a maximum of 21 days, and the medium was changed every 2 to 3 days (see FIG. 1).

[5-4] measurement of cell number, mitochondrial number and mitochondrial Activity

After completion of the culture, the culture supernatant was removed, and then the cells were washed 2 times with PBS, and a test medium (100. mu.L/well) containing 1000-fold diluted bavalomycin A1(Bafilomycin A1) was added thereto and cultured at 37 ℃ for 60 minutes. Then, the medium was replaced with a test medium (100. mu.L/well) containing Hoechst33342 solution (DOJINDO; Cat.No.346-07951, final concentration 5. mu.g/mL), 10. mu.g/mL Vectacell Rhodamine 123 (FUNAKOSHI; Cat.No. CB-2100), and 500nM MitoTracker Red CM-H2Xros (Invitrogen; M7512), and CO was added for 30 minutes₂Incubator (5% CO)₂After reaction at 37 ℃ C., the staining solution was removed from each well, washed 2 times with PBS, and then the test medium was added again, and the fluorescence intensity value of each well was measured with a microplate reader. The total number of cells was calculated from the intensity of fluorescence when they were stained with Hoechst (Hoechst), and the relative fluorescence values were calculated by averaging the intensity of fluorescence when the Vectacell Rhodamine 123 stain and the MitoTracker Red CM-H2Xros stain were stained with the Hoechst value. The measurement wavelengths are shown below.

Hoechst33342 (cell number):

340nm (excitation wavelength)/461 nm (fluorescence wavelength)

MitoTracker Red CM-H2Xros (mitochondria):

579nm (excitation wavelength)/599 nm (fluorescence wavelength)

rhodamine 123 (mitochondrial activity):

505nm (excitation wavelength)/534 nm (fluorescence wavelength)

[5-5] determination of type I collagen amount

After each culture, the culture supernatant (collagen accumulation after 3 days of each culture) was collected and stored at-80 ℃ until measurement. The stock solution was thawed on ice at the time of measurement, and the measurement was carried out using a human type I collagen ELISA kit (ACEL Co., Ltd.; EC 1-E105). The amount of type I collagen was measured as follows according to the instructions attached to the kit.

Based on the collagen standard stock solution (200. mu.g/mL), 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625. mu.g/mL collagen standard solutions were prepared using a reaction buffer, and the collagen standard solution, the specimen (culture supernatant), and the biotin-labeled collagen antibody solution were mixed in a new tube so that the ratio was 9: 1. The collagen-immobilized microplate was washed 3 times with a washing solution, and a mixed solution of a collagen standard solution, a specimen (culture supernatant), and a biotin-labeled collagen antibody solution was added to each well at 50. mu.L, followed by a reaction at room temperature for 60 minutes while shaking with a plate mixer (primary reaction). Then, the solution in the wells was removed, washed 3 times with a washing solution, and a Horseradish peroxidase (HRP) -labeled antibiotic solution was added to each well at 50 μ L per well, and reacted at room temperature for 60 minutes while shaking with a plate mixer (secondary reaction). Next, the solution in the wells was removed, washed 3 times with a washing solution, 50. mu.L of the enzyme substrate solution was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes in the dark (color reaction). Then, 50. mu.L of the reaction-stopping solution was added to each well, and after mixing for 1 minute with a plate mixer, the absorbance (measurement wavelength: 450nm) of each well was measured with a microplate reader, and the collagen concentration in the sample was calculated from the standard curve.

[5-6] results

The results of measuring the number of cells, the number of mitochondria, the activity of mitochondria and the amount of type I collagen are shown in FIGS. 2 to 5. As for the number of cells, as shown in FIG. 2, no difference was observed between each test substance and each concentration region. From this result, it was found that the cell growth promoting properties of each test substance were not observed. As for the number of mitochondria, as shown in fig. 3, a significant increase in the number of mitochondria was observed in the horse placenta hydrolysate-supplemented area at the time of 3 days of culture, but the effect of increasing the number of mitochondria by the addition of horse placenta hydrolysate was not observed in the long-term treatment after 7 days of treatment. On the other hand, no mitochondrial number increasing effect was seen in the low molecular weight high-grade gelatin derived from pig and the medium molecular weight high-grade gelatin derived from pig. From this, it was found that the number of mitochondria increased in a short period in normal human fibroblasts (human cells) in the treatment of adding the horse placenta hydrolysate.

next, mitochondrial activity was evaluated, as shown in fig. 4, in a long-term treatment after 7 days of culture, the mitochondrial activity was significantly enhanced by the treatment with horse placenta hydrolysate, but no enhancement in mitochondrial activity was seen in the low molecular weight high-grade gelatin derived from swine and the medium molecular weight high-grade gelatin derived from swine. Further, resveratrol used as a positive control for mitochondrial activation showed only a mitochondrial activity-enhancing effect in 21 days of culture treatment. From this, it was found that horse placenta hydrolysate enhances the mitochondrial activity of normal human fibroblasts (human cells). In addition, as for the evaluation of the type I collagen production, as shown in fig. 5, at 14 days and 21 days of culture, the production of type I collagen was increased by the treatment with the horse placenta hydrolysate, but the increase of collagen was not observed in the low molecular weight high-grade gelatin derived from swine and the medium molecular weight high-grade gelatin derived from swine, and needless to say, a partial decrease was observed. Vitamin C (ascorbic acid), used as a positive control for promotion of type I collagen production, showed a significant type I collagen increasing effect in all culture zones. Since collagen in each test substance is likely to be detected as type I collagen, collagen in a test medium containing the test substance in advance was measured, but type I collagen was not detected in all media. From this, it is known that horse placenta hydrolysate promotes the production of type I collagen in normal human fibroblasts (human cells).

From the above, it can be seen that: horse placenta hydrolysate has effects of increasing mitochondria number in normal human fibroblast (human cell), enhancing mitochondria activity, and promoting collagen production. In addition, it is also known that: horse placenta hydrolysate increased mitochondrial number in the short term and increased mitochondrial activity in the long term. The promotion of type I collagen production is considered to be accompanied by the activity of mitochondria in normal human fibroblasts (human cells), and the enhancement of type I collagen production ability in human fibroblasts. Since the number of cells per se does not change during each culture period, it is considered that the action of horse placenta hydrolysate to increase the number of mitochondria in normal human fibroblasts (human cells), enhance the activity of mitochondria, and promote the production of collagen is not an action accompanying cell proliferation but an action to promote the cell function per se.

6. cell migration test by the scratch method

In the process of wound healing, it is known that normal human fibroblasts (human cells) proliferate and migrate to the wound site to contract and seal the wound, and the cell migration promoting effect by horse placenta hydrolysate is verified. Specific test methods are as follows.

[6-1] test method for cell migration test

Cross lines were drawn in advance on the back side of a 60mm Dish (Dish), in this case 4000/cm²The normal human fibroblasts (human cells) are seeded at the ratio of (a) above (b), cultured until the cells become confluent, and then the cells in the confluent state are peeled off by a dedicated bar and scratched. Next, the culture medium to which the horse placenta hydrolysate sample of the present example was added or the culture medium to which the sample was not added was exchanged, and images were taken after 0 hour, 24 hours, and 48 hours, and the distance into the blank portion was measured with the cross line due to the scratch as a reference. Where 2 and 3 sites were measured and averaged in 1 well, and the number of n was set to 4 or 5. The results are shown in FIG. 6.

[6-2] results

As shown in fig. 6, in the case of adding the horse placenta hydrolysate used in the present example (100 μ g/mL), migration activity was improved together with proliferation of normal human fibroblasts (human cells) with the passage of time of 0 hour, 24 hours, and 48 hours, as compared with the case of not adding the horse placenta hydrolysate. From this, it can be seen that the horse placenta hydrolysate used in the present example has wound healing ability. As described above, since growth factors such as EGF (epithelial cell growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor), and the like, which are conventionally known to be contained in placenta extracts, are proteins and are decomposed and inactivated by hydrolysis reaction, the wound healing agent of the present invention can be said to have a different wound healing effect from that of the conventional placenta extracts.

7. skin proliferation ability confirmation test

In order to confirm whether or not the horse placenta hydrolysate has the skin proliferation ability, an experiment was performed using a three-dimensional culture kit for epidermal tissue. Specific test methods are as follows. Wherein the operation is performed in a clean bench, and sterilized instruments are used as the instruments.

[7-1] test method for skin growth ability confirmation test

This test was carried out using a human epidermis modeling KIT (LabCyte EPI-KIT; Japan Tissue Engineering Co., Ltd.) according to the instructions described therein.

[7-2] results

Fig. 7 shows the culture process (day 1 → day 4 → day 7 → day 10 → day 14 → day 18 → days 21 and 28) in the growth example of the human epidermal MODEL, fig. 8(a) shows the comparison between the cross section of the human epidermal MODEL (LabCyte EPI-MODEL) and the cross section of the human normal skin (eyelid), fig. 8(b) shows the configuration and state of subculture in the three-dimensional culture kit, fig. 9 shows the test results ((a) shows the comparison, (b) shows the case where 5% (w/w) of the horse placenta hydrolysate is added, and (c) shows the case where 50% (w/w) of the horse placenta hydrolysate is added). As shown in fig. 9, although no epidermal tissue was formed when normal human fibroblasts (human cells) were subcultured on a medium to which horse placenta hydrolysate used in the present example was not added (fig. 9(a)), epidermal tissue was formed and proliferated when normal human fibroblasts (human cells) were subcultured on a medium to which horse placenta hydrolysate used in the present example was added (fig. 9(b) and (c)). From this, it was found that the horse placenta hydrolysate used in the present example had skin proliferating ability.

8. Skin irritation safety test

In order to confirm the skin irritation of horse placenta hydrolysate, experiments were performed using a three-dimensional culture kit for epidermal tissue and a measurement kit for skin irritation. Specific test methods are as follows.

[8-1] test method for skin irritation test

The test was carried out using CellTiter96Non-Radioactive CellProlification Assay (Promega) as a human epidermis modeling KIT (LabCyte EPI-KIT; Japan Tissue Engineering) and a skin irritation measurement KIT according to the instruction manual for LabCyte EPI-KIT and the description of "skin irritation test method by human three-dimensional culture epidermis EPI-MODEL 24". The skin irritation was measured according to the following procedure.

First, a human epidermal model was prepared in the same manner as in the "test for confirming skin proliferation ability" 7. Next, as positive control substances, 5% (w/w) Sodium Lauryl Sulfate (SLS) as a skin irritant, 50% (w/w) horse placenta hydrolysate, and 5% (w/w) horse placenta hydrolysate were used as samples, and each sample was brought into contact with the prepared human epidermis model, cultured for 5 days, and then each sample was removed, and cytotoxicity was measured by the MTT method. To MTT medium (2mL or 3mL) cultured in a 60mm dish was added 100. mu.L of Dye Solution (Dye Solution) and the mixture was incubated at 37 ℃ in CO₂After 4 hours of incubation in an incubator, 1mL of a Solution/Stop Mix was added and incubated at 37 ℃ in CO₂Culturing in an incubator for 1 hour. Then, 200. mu.L of each was transferred to a well of a 96-well plate, and then absorbance at 560nm was measured with a microplate reader.

[8-2] results

The results of this test are shown in fig. 10. In fig. 10, (a) shows the results when 5% SLS was used as a sample, (b) shows the results when 50% (w/w) of the horse placenta hydrolysate was used as a sample, and (c) shows the results when 5% (w/w) of the horse placenta hydrolysate was used as a sample. As shown in fig. 10, when 5% SLS was used as a sample, the number of cells became about 35%, which caused damage to the human epidermal model; however, when 50% (w/w) of horse placenta hydrolysate was used as a sample, the number of cells was about 115%, which was increased by about 15%; when 5% (w/w) of horse placenta hydrolysate was used as a sample, the number of cells was about 108%, which was increased by about 8%. From this, it is understood that the horse placenta hydrolysate used in the present example is highly safe to the skin, and can be said to promote the growth of each tissue and cell of the skin.

Claims

1. A formulation, characterized by:

Which comprises 1 or 2 or more horse placenta hydrolysates selected from the following (a), (b) and (c) as an active ingredient,

(a) A mitochondrial activator,

(b) I type collagen production promoter,

(c) A wound healing agent.

2. a formulation, characterized by:

It is a preparation containing horse placenta hydrolysate as effective component, and has effects in increasing mitochondria amount in human cells in short term and enhancing mitochondria activity in long term.

3. A formulation, characterized by:

It is a wound healing agent containing horse placenta hydrolysate as an active ingredient and healing wounds by proliferating human cells and improving their migration activity.

4. The formulation according to any one of claims 1 to 3, wherein:

It is horse placenta hydrolysate hydrolyzed by cysteine protease derived from fruit.

5. The formulation according to any one of claims 1 to 4, wherein:

Horse placenta hydrolysate contains most peptides as a constituent.

6. The formulation of claim 5, wherein:

Horse placenta hydrolysate contains peptide with molecular weight of 1000 or more and less than 3000 as the most.

7. A skin care cosmetic characterized by:

Which contains the preparation according to any one of claims 1 to 6, a humectant and a preservative.