KR101414150B1 - Cosmetic compositions for anti-wrinkle effect comprising hydrolyzed placenta extract using high pressure system - Google Patents

Abstract

The present invention relates to an anti-wrinkle cosmetic composition comprising a low-molecular placenta extract hydrolyzed by an ultra high-pressure system. Since the composition of the present invention contains a low-molecular placenta extract having a molecular weight of 100 to 1,500 Da obtained by hydrolyzing the placenta at a pressure of 10 to 500 bar and a temperature of 120 to 250 ° C for 20 minutes to 3 hours, . In addition, anti-wrinkle effect can be seen by inhibiting the activity of MMP-2 and elastase.

Description

[0001] The present invention relates to an anti-wrinkle cosmetic composition comprising placenta extract hydrolyzed by an ultra high pressure system,

More specifically, the present invention relates to a cosmetic composition for anti-wrinkles. Specifically, a placenta extract having low molecular weight can be obtained by hydrolyzing the placenta in an ultrahigh pressure system, and when a cosmetic composition containing a low-molecular placenta extract is used, The present invention relates to a cosmetic composition for anti-wrinkles containing placenta extract hydrolyzed by an ultra high-pressure system having an anti-wrinkle effect and an anti-wrinkle effect by inhibiting the activity of elastase and MMP-2.

Wrinkles are caused by various factors inside and outside of the body, such as collagen fibers in the dermis, elastic fibers and denature occurs, and skin moisture is reduced, causing the skin to lose its elasticity and folding the skin. In recent years, there has been an increasing interest in prevention and improvement of skin wrinkles as the level of living has improved and interest in the appearance has increased.

The structure of the skin can be divided into two layers, epidermis and dermis. The epidermis is a thin protective layer which is located in the uppermost layer of the skin and is composed of keratinocytes, melanocytes, and the like. And the dermis is a thick and complex layer of fibrous tissue and connective tissue in which elastic fibers such as collagen and elastin form a network structure.

Collagen in the dermis is the main structural protein of the skin, which accounts for about 70% of the dermis. It is a fiber structure that gives the skin the flexibility and protects the skin from external stimuli or forces by giving strength and tension to the skin. Therefore, when decomposition of collagen proceeds or collagen synthesis is reduced, the wrinkles become deeper and the elasticity decreases, and the skin becomes sagged or the surface becomes rough.

Specifically, the polypeptide chain of collagen is synthesized in a state of having a telopeptide at both ends in a granule cell wall of fibroblasts, and is secreted out of the cell in the form of procollagen combined with a triple helix structure . The secreted pro-collagen is cleaved by enzymes to produce tropocollagen, which is then combined with collagen in the extracellular matrix. However, the synthesis of collagen rapidly decreases as the skin ages, and collagen is deformed by the external environment, resulting in wrinkles on the skin.

Examples of enzymes associated with skin wrinkles include collagenase, matrix metalloproteinase (MMP), elastase, and the like. Among them, collagenase increases expression and activity by external factors such as ultraviolet rays, heat or oxidative stress, and internal factors such as skin aging, and thereby, collagen is decomposed to generate wrinkles.

In addition, matrix metalloproteinase (MMP) plays an important role in skin photoaging by collapsing the collagen of the dermis in association with the degradation of the extracellular matrix (ECM) and the basement membrane (BM).

In addition, elastase degrades elastin associated with skin elasticity, thereby breaking the binding of the network structure formed by collagen and elastin, thereby affecting the formation of wrinkles in the skin.

On the other hand, the placenta is a structure that mediates the exchange of substances necessary for the survival and growth of the fetus between the fetus and the mother. The placenta is formed by adhering part of the placenta surrounding the fetus to the endometrium of the mother. The placenta supplies oxygen and various nutrients to the fetus and not only discharges the waste generated by the fetus, it plays a variety of roles such as generating hormones and various factors required for the fetus.

The placenta also contains various amino acids, vitamins, active peptides, minerals, cytokines, growth factors such as insulin-like growth factor (IGF), hepatocyte growth factor (HGF) and fibroblast growth factor Because it contains a large amount of ingredients, it can perform various actions. Thus, the placenta is known to be effective in improving immunity or restoring fatigue.

In particular, the placenta of the pig is very similar to the molecular structure of the placenta in the mammalian placenta. It is known to be relatively safe, with the advantage of being easily recovered and securing a large amount of placenta.

Therefore, attempts to utilize the placenta in various fields such as cosmetics, health functional foods, medicines, and the like are continuing. For example, many products such as cosmetics containing placenta extract are sold.

However, when the placenta extract is used as a cosmetic to be applied to the skin, there is a disadvantage that it is difficult to absorb the effect of moisturizing action because the placenta extract is a polymer and it is difficult to absorb on the skin. In the case of the polymer placenta extract, There is a difficulty in manufacturing and using.

Thus, since the placenta extract itself is a polymer, its use is limited. Therefore, various methods for producing a placenta extract having a small molecular weight have been proposed in order to increase the utilization efficiency of the placenta extract.

However, when the placenta protein is degraded by using a strong acid such as hydrochloric acid or a strong alkaline substance, the active ingredient may lose its activity during the high temperature treatment for a long period of time. Especially, when strong acid is used, There is a problem that additional equipment cost is incurred.

When protease such as pepsin, trypsin, and chymotrypsin is used, it takes a long time for low molecular weight of placental protein and there is a restriction of the reaction temperature such as reaction at the highest enzyme activity. When the temperature is exceeded, there is a problem that the decomposition efficiency is lowered or the enzyme is inactivated.

Thus, the conventional methods have a problem in that it takes a long time to decompose the placental protein and the active ingredient loses its activity in the extraction process. Therefore, a method of low molecular weight of the placenta protein having excellent extraction efficiency without losing the active ingredient Is required.

Regarding the cosmetic composition containing the placenta extract, Korean Patent Laid-Open Publication No. 10-2008-0096219 discloses a cosmetic composition comprising a placenta extract hydrolyzed by treatment with a protease, 0109391 discloses a wrinkle-improving and whitening cosmetic composition comprising a porcine placenta extract obtained by lyophilizing a porcine placenta and then hydrolyzing it with alkali and adjusting its pH. Korean Patent Publication No. 10-2011-0134146 discloses And an external preparation for skin whitening comprising at least one of an extract obtained by hydrolysis with a protease and horse oil.

However, no anti-wrinkle cosmetic composition containing a low-molecular placenta extract prepared by hydrolyzing the placenta using an ultra-high pressure system as in the present invention has been disclosed.

Accordingly, the present inventors have made efforts to develop a cosmetic composition containing a low-molecular placenta extract. As a result, they have found that when hydrolyzing placentae under ultrahigh pressure conditions, they do not use strong acids or strong bases, Therefore, the present inventors have found that the cosmetic composition containing the same has excellent effects when it is used, and completed the present invention.

One object of the present invention is to clean the placenta, remove the foreign matter, cut into a size of 1 to 50 mm and then dipped in purified water at 3 to 6 캜 for 12 to 24 hours to remove blood, A low molecular weight placenta extract prepared by diluting with 1.5 to 3 parts by weight of distilled water and homogenizing for 5 to 30 seconds and then hydrolyzing at a pressure of 10 to 500 bar and a temperature of 120 to 250 ° C for 20 minutes to 3 hours To provide a cosmetic composition for anti-wrinkles.

Another object of the present invention is to provide a cosmetic composition for anti-wrinkles, wherein the low molecular weight placenta extract has a molecular weight of 100 to 1,500 Da.

In one embodiment, the present invention relates to a method for treating a placenta, which comprises washing the placenta, removing foreign matter, finely dividing the blood into a size of 1 to 50 mm, and then immersing in purified water at 3 to 6 캜 for 12 to 24 hours to remove blood, A low molecular weight placenta extract prepared by diluting with 1.5 to 3 parts by weight of distilled water and homogenizing for 5 to 30 seconds and then hydrolyzing at a pressure of 10 to 500 bar and a temperature of 120 to 250 ° C for 20 minutes to 3 hours, The present invention relates to a cosmetic composition for anti-wrinkles.

The placenta of the present invention mediates the exchange of substances necessary for the survival and growth of the fetus between the fetus and the mother. The placenta can be thawed after the placenta collection, or it can be used after thawing. May be used.

The placenta is not limited to use on the placenta of a mammal such as a human, a monkey, a sheep, a cattle, a dog, or a pig, but is preferably a placenta of a pig. The porcine placenta has characteristics similar to those of the human placenta, and is easy to recover, secures a large amount, and contains various nutrients including proteins.

Hereinafter, a method for producing the anti-wrinkle cosmetic composition containing the low-molecular placenta extract will be described in detail.

First wash the placenta with water or physiological saline. The above washing is repeatedly carried out until cleaned water or physiological saline is used to remove foreign materials attached to the placenta including the meconium, until the foreign substance which is visually confirmed disappears. If the foreign matter is not removed cleanly, the produced placenta extract may contain impurities, which may deteriorate the quality.

Thereafter, the washed placenta is cut into a size of 5 to 50 mm. If the size of the chopped placenta is less than 5 mm, it takes a long time for the chopping, which is inefficient. If the size of the chopped placenta exceeds 50 mm, the blood contained in the placenta is not sufficiently removed and the quality of the prepared placenta extract may be deteriorated. And, the method is not limited if it makes the placenta small in size, for example, it can be cut using the esophagus.

Next, the chopped placenta is immersed in purified water at 3 to 6 캜 for 12 to 24 hours to remove it. If the temperature of the purified water is less than 3 ° C, the placenta may partially freeze and the blood may not be sufficiently removed. If the temperature exceeds 6 ° C, denaturation of the placenta protein may occur, or blood may not be well absorbed into the purified water. The temperature of the purified water is preferably 3 to 6 占 폚. In addition, in the case of using the purified water in which the blood of the placenta is continuously used during the immersion process, it is difficult to confirm the condition of the placenta and blood may not be sufficiently removed, so it is preferable to change the purified water to be used at any time.

Thereafter, the washed placenta is dehydrated to remove water, and then pulverized using a pulverizer. The pulverization is performed so as to increase the extraction efficiency of the placenta extract, which is a subsequent process, so that the placenta is sufficiently small and uniform in size.

Next, the pulverized placenta is centrifuged to obtain a precipitate in which the water of the placenta is completely removed, and 1.5 to 3 parts by weight of distilled water is added to the precipitated placenta to dilute the placenta in distilled water.

The diluted placenta is then homogenized. The homogenization is a process of evenly dispersing the placenta in distilled water, which can be performed using a homogenizer or a blender. The homogenization time is not limited as long as the placenta can be sufficiently dispersed in the distilled water, but homogenization is preferably performed for 5 to 30 seconds. When the homogenization time is less than 5 seconds or more than 30 seconds, the recovery rate of the placenta in the subsequent hydrolysis process may be lowered, the molecular weight may not be sufficiently decreased, and the effect is similar to that of homogenization for 5 to 30 seconds, which is inefficient .

Next, the homogeneous material is hydrolyzed using an ultra-high pressure system at a pressure of 10 to 500 bar and a temperature of 120 to 250 ° C for 20 minutes to 3 hours.

The hydrolysis is preferably carried out in a subcritical state using an ultrahigh pressure system. The subcritical state refers to a superheated region having a lower temperature and pressure than a critical point of the solution. In the case of carrying out the hydrolysis in the subcritical state, the extract can be rapidly obtained as compared with the general case, and since the extraction efficiency is excellent even without using an acid or base as a solvent, there is no fear that the active ingredient is destroyed by an acid or a base. Therefore, when the solvent is used as water, hydrolysis is preferably carried out under the conditions of a pressure of 10 to 500 bar and a temperature of 120 to 250 ° C.

When the pressure is less than 10 bar, the placenta protein is not decomposed sufficiently, resulting in a high molecular weight. Thus, the use of the placenta extract produced may lower the absorption efficiency of the skin, resulting in a decrease in the use efficiency. . Therefore, the pressure for hydrolysis is from 10 to 500 bar, preferably from 100 to 500 bar, more preferably from 200 to 500 bar, and most preferably from 300 to 400 bar.

When the temperature is less than 120 ° C, the placenta protein is not sufficiently decomposed, resulting in a high molecular weight. Thus, the placenta extract produced may have a low absorption rate of the skin, resulting in a decrease in utilization efficiency. When the temperature exceeds 250 ° C, The recovery rate of the low-molecular-weight placenta extract produced by hydrolysis may be lowered. Therefore, the temperature for hydrolysis is preferably 120 to 250 ° C.

If the hydrolysis time is less than 20 minutes, the hydrolysis does not occur sufficiently, and the molecular weight of the placenta protein is so large that the efficiency of the prepared placenta extract tends to be lowered. If the hydrolysis time exceeds 3 hours, the placenta extract May not exhibit its effect, it is preferable that the hydrolysis time is 20 minutes to 3 hours.

When the placenta extract is prepared for 20 minutes to 3 hours at a pressure of 10 to 500 bar and a temperature of 120 to 250 ° C as described above, a placenta extract having a small molecular weight can be obtained.

The molecular weight is 100 to 1,500 Da, preferably 200 to 1,200 Da, more preferably 300 to 1,000 Da.

Since the low molecular weight placenta extract prepared by hydrolysis under the high pressure condition does not use organic acid in the course of low molecular weight of placental protein, the active ingredient is not denatured, and the production time is shortened compared with the conventional method, The low-molecular-weight placenta extracts produced are economical because they have a high recovery rate.

Meanwhile, the low-molecular placenta extract prepared by the above-mentioned method can be used as a cosmetic composition for anti-wrinkles.

When the low-molecular-weight placenta extract is used as a cosmetic composition, it inhibits the activity of elastase and MMP-2 and inhibits cytotoxicity caused by UVB, thereby preventing and improving wrinkles of the skin. Especially, the low molecular weight placenta extract has a higher absorption rate of the skin than the low - molecular - weight placenta protein, so that it can exhibit excellent anti - wrinkle effect even when a small amount is used.

In one specific embodiment, hydrolysis of the placenta using ultrahigh pressure increased the skin uptake rate compared to the non-hydrolysed placenta extract, and the skin uptake increased in a concentration-dependent manner in the placenta extract. It was confirmed that the utilization efficiency was good.

The low molecular weight placenta extract of the present invention is contained in the composition in an amount of 0.00001 to 15% by weight, preferably 0.0001 to 10% by weight, and most preferably 0.0001 to 5% by weight based on the total weight of the composition. When the weight of the low-molecular-weight placenta extract is less than 0.00001% by weight, the anti-wrinkle effect is weak. When the content is more than 15% by weight, .

On the other hand, the cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the low molecular weight placenta extract as an active ingredient having anti-wrinkle effect. Such as antioxidants, stabilizers, solubilizers, vitamins, customary adjuvants such as pigments and flavoring agents, and carriers.

The cosmetic composition may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing oil, powder foundation, emulsion foundation, wax foundation, spray, Lotion, essence, nutritional gel or massage cream, but is not limited thereto.

When the formulation of the cosmetic composition is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

When the formulation of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of spray, a mixture of chlorofluorohydrocarbons, Propane / butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , Propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan, and the like.

When the formulation of the cosmetic composition is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the cosmetic composition is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

Particularly, the cosmetic composition comprising the low-molecular placenta extract prepared as described above is safe because it does not use harmful strong acids or strong bases during its production, and because the placenta extract has a small molecular weight and excellent skin absorption rate, Effect can be obtained.

As described above, the cosmetic composition containing the low-molecular placenta extract of the present invention can inhibit the activity of elastase and MMP-2 and inhibit cytotoxicity caused by UVB to obtain anti-wrinkle effect. Particularly, since the low molecular weight placenta extract has a high skin absorption rate, excellent anti-wrinkle effect can be obtained even when a small amount of the cosmetic composition is used as compared with a cosmetic composition containing a placenta extract without a low molecular weight.

FIG. 1 is a graph showing changes in the content of proteins according to the hydrolysis time during hydrolysis of the placental protein at the ultrahigh pressure of the present invention.
FIG. 2 is a graph showing changes in the content of amino acids according to the hydrolysis time during hydrolysis of the placenta protein at ultra-high pressure of the present invention.
FIG. 3 shows elastase activity according to hydrolysis time at hydrolysis of the placenta protein at ultra high pressure of the present invention.
FIG. 4 shows the skin absorption rate according to the hydrolysis time during the hydrolysis of the placenta protein at the ultrahigh pressure of the present invention.
FIG. 5 shows the expression level of MMP-2 according to the concentration upon hydrolysis of the placenta protein of the present invention. Concentrations of UVB and UVB were measured in the control, ATRA, and NETC, respectively. The placental protein was hydrolyzed for 3 hours at ultra high pressure. The ETC was hydrolyzed for 3 hours at ultra - .
FIG. 6 shows the degree of MMP-2 expression according to the concentration upon hydrolysis of the placenta protein of the present invention. Concentrations of UVB and UVB were measured in the control, ATRA, and NETC, respectively. The placental protein was hydrolyzed for 3 hours at ultra high pressure. The ETC was hydrolyzed for 3 hours at ultra - .

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example 1: Preparation of low-molecular placenta extract using ultrahigh pressure

1-1. Collection of pig placenta

The placenta to be used for the experiment was collected from the livestock of Samwoo Livestock which collected the placenta which the pigs discharged at the time of delivery.

1-2. Washing of pig placenta

The porcine placenta collected in Example 1-1 was thawed using a thawing machine, and the foreign matter including meconium was removed, followed by washing with physiological saline. Thereafter, the placenta washed in the above was cut to a size of 5 to 50 mm, and then immersed in purified water at 3 to 6 캜 for 12 to 24 hours to remove blood.

1-3. Production of low molecular placenta extract

The placenta washed in Example 1-2 was dehydrated, the water was removed, pulverized using a pulverizer, and 1.5 to 3 parts by weight of distilled water was added to the placenta to dilute it. The placenta was homogenized for 5 to 30 seconds, 300 to 400 bar and 120 to 250 ° C for 20 minutes to 3 hours to obtain a low molecular weight placenta extract.

Example  2: Protein content analysis of hydrolyzed placenta extract using ultrahigh pressure

In order to analyze the change of protein content according to the hydrolysis time during hydrolysis of the placenta protein at ultra-high pressure, the homogenized placenta of Example 1-3 was incubated at 0, 0.5, 1, and 2 hours After the digestion, the content of protein was quantified using Thermo's BCA ^™ protein assay kit. First, 25 μl of the placenta extract was mixed with 200 μl of BCA working solution and reacted at 37 ° C. for 30 minutes. Thereafter, the absorbance at 562 nm was measured using a spectrophotometer, and the results are shown in FIG.

The change of amino acid content in hydrolysis of placental protein at high pressure was measured by Alder-Nissen's TNBS method. First, 50 μl of the placenta extract was mixed with 400 μl of sodium phosphate buffer and 0.1% TNBS, followed by reaction for one hour with blocking of light to develop color. Then, 800 μl of 0.1 N hydrochloric acid and 1.6 ml of distilled water were added to the reaction product, and the absorbance at 340 nm was measured using a spectrophotometer. The result is shown in FIG.

As a result of the experiment, BCA content decreased as the hydrolysis time increased, but the total AN content was increased. As a result, the protein was changed to amino acid due to hydrolysis.

Example 3 Evaluation of Elastase Inhibitory Activity of Hydrolyzed Placenta Extract Using Ultra High Pressure

To analyze elastase activity according to the hydrolysis time during hydrolysis of placental protein at ultra-high pressure, 1 쨉 g of porcine pancreatic elastase (PPE) type IV was dissolved in 0.2M Tris-HCl Dissolved in 1 ml of buffer. Thereafter, a mixture of 0.2 M Tris-HCl buffer, 1 ppm of porcine pancreas-derived elastase and 0.8 mM N- succinyl- (Ala) ₃ - p- nitoanilide was added at 0, 0.5, The decomposed placenta extracts were mixed and reacted. Next, the absorbance of the reactant was measured at 410 nm using a spectrophotometer, and the inhibition rate of the elastase activity of the placenta extract was calculated using the following Equation 1, and IC ₅₀ was measured. The results are shown in Tables 1 and 3 Respectively. IC ₅₀ refers to a concentration at which the activity of the elastase is reduced by 50% compared to the initial level.

[Experimental Equation 1]

Inhibition rate = [(A-B) - (C-D)] / (A-B) * 100

B means the absorbance in the absence of both the sample and the elastase, C means the absorbance in the case where both the sample and the elastase are present, D Means the absorbance when there is only sample without elastase.

As a result of the experiment, it was observed that the IC ₅₀ value of the placenta extract tended to decrease as the hydrolysis time increased, and the elastase inhibitory effect was excellent. However, when the hydrolysis time was longer than 2 hours, .

Example 4: Evaluation of skin absorption rate of hydrolyzed placenta extract using ultrahigh pressure

To investigate the skin absorption rate according to hydrolysis time during hydrolysis of placental protein at ultra-high pressure, the skin of 8-week-old male SD rats was peeled off and cut to a size of 1.5 cm ² , followed by Franz-type diffusion cell ) As a membrane. Then, 4.9 ml of a 0.1M sodium phosphate buffer solution of pH 7.4 was added to the receptor portion, and the placenta extract hydrolyzed at 0, 0.5, 2 and 3 hours at 300 bar and 200 ° C under ultrahigh pressure was applied to a donor ). Next, the sodium phosphate buffer solution was stirred at a speed of 400 x g while maintaining the temperature of the Franz-type diffusion mechanism at 37 占 폚, and at the time of 0.5, 1, 2, 3, 4, 0.5 ml of a sodium phosphate buffer solution was added, and 0.5 ml of a fresh sodium phosphate buffer solution was added. The content of the hydrolyzed placenta protein in the solution obtained above was measured to examine the skin absorption rate of the hydrolyzed placenta extract. The result is shown in FIG.

As a result, it was observed that the hydrolysis time of placenta protein increased at high pressure and that the skin absorption rate increased with increasing agitation time.

Example 5 Evaluation of Skin Protection Effect by UVB of Hydrolyzed Placenta Extract Using Ultra High Pressure

5-1. Cytotoxicity Assessment

To evaluate cytotoxicity upon hydrolysis of placental proteins at ultra-high pressure, HaCaT cells, 2 x 10 ⁵ human-derived keratinocytes per 1 ml of DMEM medium, were dispensed into 96-well plates. After 24 hours, the placenta extract without control, hydrolyzed placenta extract at 300 bar and 200 ° C for 3 hours, 300 bar of ultrahigh pressure, and placenta hydrolyzed at 200 ° C for 3 hours were again digested with enzyme Placenta extracts were treated with cells at concentrations of 0, 1, 10, 50, 100 and 250 占 퐂 / ml, and MTT assay was performed 24 hours later to evaluate cell viability of each group. Respectively.

As a result of the experiment, the placenta extract without digesting enzymes did not hydrolyze and the control group showed cytotoxicity at the concentration of 10 ㎍ / ㎖, and only about half of the cells survived at 100 ㎍ / ㎖. In the case of placenta extract treated with digestive enzymes after hydrolysis, cytotoxicity was observed from 250 μg / ㎖. However, the hydrolyzed placenta extract showed no cytotoxicity even at the highest concentration of 250 μg / ml.

5-2. Evaluation of inhibition of cell damage by UVB

To investigate the degree of inhibition of UVB-induced cell damage by hydrolysis of placental proteins at ultra-high pressure, 2 X 10 ⁵ HaCaT cells per 1 ml of DMEM medium were dispensed into 96-well plates. After 24 hours, the placenta extract without control, hydrolyzed placenta extract at 300 bar and 200 ° C for 3 hours, 300 bar of ultrahigh pressure, and placenta hydrolyzed at 200 ° C for 3 hours were again digested with enzyme Placenta extracts were treated with cells at 0, 1, 10, 25, 50 and 100 ㎍ / ㎖. After 30 minutes, the cells were irradiated with UVB at 15 mJ / cm ² for 1 minute and 30 seconds at an intensity of 160 ㎼, and MTT assay was performed 24 hours later. As compared with the cells not treated with UVB, the cell survival (cell viability) is shown in Table 3 below.

As a result, the control group, which was a placenta extract without digesting enzyme without hydrolysis, exhibited cytotoxicity at a concentration of 10 μg / ml and showed no effect to protect cells damaged by UVB. However, in the case of hydrolyzed placenta extract and placenta extract treated with digestion enzyme after hydrolysis, the cell survival rate of each sample treated with UVB tended to be slightly increased, which showed a cytoprotective effect.

Example 6: Evaluation of MMP-2 activity of hydrolyzed placenta extract using ultrahigh pressure

In order to test the degree of MMP-2 expression according to the concentration upon hydrolysis of the placental protein at ultra-high pressure, 2 X 10 ⁵ HaCaT cells per 1 ml of DMEM medium were divided into 96-well plates. After 24 hours, the placenta extract without hydrolysis, the placenta extract hydrolyzed at 200 bar and the high pressure of 300 bar for 3 hours, the placenta hydrolyzed at 300 bar and 300 bar for 3 hours, were digested with digestion enzyme again Each of the placenta extracts was treated with 1, 10, 25, 50 and 100 ㎍ / ㎖ of the cells and irradiated with UVB at 15 mJ / cm ² for 1 minute and 30 seconds at an intensity of 160 30 after 30 minutes. Twenty-four hours later, the RNA was isolated from the cells using the TRIzol LS reagent. RT-PCR was performed using the RevertAid ^™ First Strand cDNA Synthesis Kit, followed by 1% agarose gel ), And the degree of expression of MMP-2 was confirmed. The results are shown in FIG. 5 and FIG. ATRA (all-transretinoic acid) was used as a positive control.

As a result, the expression of MMP-2 was increased by about 170% when UVB was irradiated. However, the placental extract hydrolyzed at 300 bar and 200 ° C for 3 hours, the placenta treated with 300 bar and 3 hours hydrolysis at 200 ° C, and the placenta extract treated with the digestive enzymes were subjected to concentration-dependent expression of UVB-induced MMP-2 Respectively.

Claims (4)

The placenta was washed to remove foreign matter, cut into small pieces having a size of 1 to 50 mm, and then immersed in purified water at 3 to 6 ° C for 12 to 24 hours to remove blood. The placenta was then pulverized and 1.5 to 3 parts by weight of distilled water , Followed by homogenization for 5 to 30 seconds and hydrolysis at a pressure of 10 to 500 bar and a temperature of 120 to 250 ° C for 20 minutes to 3 hours to obtain an anti-placenta extract having a low molecular weight placenta extract having a molecular weight of 100 to 1,500 Da A cosmetic composition for wrinkles.
delete The cosmetic composition for anti-wrinkles according to claim 1, wherein the placenta is at least one placenta selected from pigs, human, horse, monkey and dog.
The anti-wrinkle cosmetic composition according to claim 1, wherein the low molecular weight placenta extract comprises 0.00001 to 15% by weight of the total composition.

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