KR101814744B1 - Manufacturing method of composition for inhibiting skin aging using donkey skin - Google Patents

Abstract

The present invention relates to a method of manufacturing a composition for suppressing aging by using donkey skin. More specifically, the method includes: a material pretreatment step of pretreating donkey skin; a collagen extracting step of mixing the pretreated donkey skin with water, extracting collagen at a high temperature and pressure, and filtering the mixture; a collagen decomposing step of decomposing collagen by mixing proteolysis enzymes with the collagen decomposed and extracted through the collagen decomposing step; an enzyme inactivating step of inactivating enzymes contained in the decomposed collagen; and a filtering step of filtering the decomposed collagen having the enzymes inactivated through the inactivating step. The composition for suppressing aging is capable of improving wrinkles and preventing skin aging, and minimizing stimulation to skin by omitting an alkaline or acidic treatment procedure.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for inhibiting skin aging using a donkey skin,

The present invention relates to a method for producing a composition for inhibiting skin aging using a donkey skin, and more particularly, to a method for preparing a composition for preventing aging of a skin using a donkey shell, which is less susceptible to skin irritation due to no alkali or acid treatment, And a method for producing a composition for inhibiting skin aging.

The number of elderly people aged 65 or older in Korea is steadily increasing year by year. In 2050, 37% of the total population is expected to be included in the elderly population, which is much higher than in other countries. Therefore, it is anticipated that interest in skin aging of older people as well as younger age will be heightened, and a wider range of consumers are demanding measures against skin aging.

The causes of skin aging can be classified into external factors and internal factors. Exogenous factors include daily life factors such as ultraviolet rays, environmental pollution, smoking and stress, and internal factors include age and genetic Factors. At this time, a potent mechanism for the cause of aging may be called photoaging that generates free radicals by ultraviolet rays.

In particular, the ultraviolet rays UVB causes DNA damage and reactive oxygen species (ROS), resulting in skin diseases and photoaging. The skin is an antioxidant network such as vitamin E ₁₁ and vitamin C, And protects the skin, but as the time elapses, its function gradually decreases and the skin ages.

In addition, exposure of UVB causes reduction of collagen in the skin to form wrinkles, causing pigmentation or drying and rough skin. Therefore, development of antioxidant functional material capable of inhibiting free radical generation is required to prevent skin aging , Collagenase and Elastase inhibiting function, it is required to develop a material capable of inhibiting skin diseases such as wrinkles of the skin.

Korean Patent Registration No. 10-1557409 (2015.09.25) Korean Patent Publication No. 10-2012-0063693 (June 18, 2012)

It is an object of the present invention to provide a method for producing a composition for inhibiting skin aging using a donkey shell showing less skin irritation due to no alkali or acid treatment and exhibiting excellent skin aging and wrinkle-reducing effects.

In one embodiment, the content of the disclosure includes a raw material pretreatment step for pretreating a donkey's skin, a collagen extraction filtration step in which a donkey's shell pretreated through the raw material pretreatment step is mixed with water, a high temperature and high pressure is applied to extract collagen, A collagen degradation step of decomposing collagen by mixing the proteolytic enzyme with the collagen extracted and filtered through the collagen extraction filtration step, an enzyme inactivation step of inactivating the enzyme contained in the collagen degradation product decomposed through the collagen decomposition step, And a filtration step of filtering the collagen decomposition product in which the enzyme is inactivated through the enzyme inactivation step. The method for producing the composition for inhibiting skin aging using the donkey skin is described.

Preferably, the collagen extraction step at a temperature and a 1 to a pressure of 2kgf / cm ² for mixing with the raw material pre-processing step the donkey shell of 100 parts by weight of water 150 to 250 parts by weight of pre-treatment with the 110 to 130 ℃ 50 to 70 100 parts by weight of collagen-extracted donkey shells are mixed with 80 to 120 parts by weight of water through a first extraction and filtration step of extracting and filtering the mixture at a temperature of 110 to 130 ° C and 1 to 2 kgf / ² and a second extraction filtration step of extracting and filtering at 50 to 70 minutes.

More preferably, the collagen decomposing step comprises mixing 0.05 to 0.2 parts by weight of a protease with 100 parts by weight of the collagen extracted and filtered through the collagen extraction filtration step, at a temperature of 40 to 50 ° C for 2 to 4 hours have.

More preferably, the protease can be prepared by mixing pancreatin and alkaline protease in a weight ratio of 1: 1.

Even more preferably, the enzyme inactivation step may be performed by heating the degraded collagen degraded through the collagen degradation step to a temperature of 85 to 95 ° C for 10 to 20 minutes.

Even more preferably, the filtration step may consist of a membrane filter capable of filtering out components having a molecular weight greater than 3 kDa.

The composition for inhibiting skin aging using the donkey skin as described above is a composition for inhibiting skin aging using a donkey shell showing less skin irritation and excellent skin aging resistance and anti-wrinkle effect due to no treatment with alkali or acid, .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flowchart showing a method for producing a composition for inhibiting skin aging using the disclosed donkey's skin.
FIGS. 2 to 5 are graphs showing the antioxidative activities of compositions for inhibiting skin aging using the donkey bark prepared in Example 1. FIG.
FIGS. 6 to 7 are graphs showing the wrinkle-reducing effect of the composition for inhibiting skin aging using the donkey skin prepared in Example 1. FIG.

Hereinafter, preferred embodiments of the present invention and physical properties of the respective components will be described in detail with reference to the accompanying drawings. However, the present invention is not limited thereto, And this does not mean that the technical idea and scope of the present invention are limited.

A method for preparing a composition for inhibiting skin aging using the disclosed donkey shell comprises a raw material preprocessing step (S101) for pretreating a donkey shell, a donkey shell pretreated through the raw material preprocessing step (S101), water and collagen A collagen decomposition step (S105) for decomposing collagen by mixing the proteolytic enzyme extracted and filtered through the collagen extraction filtration step (S103) (S105), the collagen decomposition step (S103) (S107) for inactivating an enzyme contained in the collagen degradation product decomposed through the enzyme inactivation step (S105) and a filtration step (S109) for filtering the collagen decomposition product in which the enzyme is inactivated through the enzyme inactivation step (S107) .

The raw material preprocessing step (S101) is a step of pretreating the donkey's skin, washing and heating the donkey's skin, and removing the fat layer.

The water is washed with water flowing through the donkey shell to remove hair and impurities remaining in the donkey shell. The heating is carried out by adding the washed donkey shell to boiling water and heating the donkey shell for 15 to 25 minutes, And removing the hair, impurities, and oil layer remaining on the substrate.

In addition, the removal of the fat layer is a step of separating the fat layer from the collagen layer by cutting the collagen layer of the donkey's skin with a boundary portion between the fat layer and the like, and washing the fat layer with water once again after the fat layer is removed.

Since the donkey bark from which the hair, impurities and fat layer are removed through the above process does not inhibit the collagen degradation due to the fat layer during the degradation process of the collagen, the collagen degradation product is obtained at a high yield can do.

The collagen extraction filtration step (S103) is a step of mixing the pretreated donkey bark with water and extracting collagen by applying high temperature and high pressure, and filtering the raw material, and more specifically, the raw material pre-treatment step S101 100 parts by weight of the donkey shell pretreated through the first extraction filtration step (150 to 250 parts by weight of water) is subjected to a first extraction filtration step of extracting and filtering at a temperature of 110 to 130 ° C and a pressure of 1 to 2 kgf / cm ² for 50 to 70 minutes 100 parts by weight of the donkey shell, from which collagen has been first extracted, are mixed again with 80 to 120 parts by weight of water through the first extraction filtration step (S103-1) and the first extraction filtration step (S103-1) and a second extraction filtration step (S103-2) of extracting and filtering at 50 to 70 minutes at a pressure of 1 to ² cm < 2 >. The first extraction filtration step (S103-1) and the second extraction filtration step (S103-2 ) Are mixed and used, When carried over the extraction process, as the group 2 times, thereby improving the extraction efficiency of the collagen contained in the skin of a donkey.

It is preferable that the filter used in the first extraction filtration step (S103-1) and the second extraction filtration step (S103-2) be a filter capable of filtering particles of 100 to 200 micrometers.

Through the collagen extraction filtration step (S103) comprising the above process, the collagen components contained in the donkey bark extract can be extracted at low cost, excellent yield, and short processing time.

The collagen decomposition step (S105) is a step of decomposing collagen by mixing the proteolytic enzyme with the collagen extracted and filtered through the collagen extraction filtration step (S103). The collagen extraction filtration step (S103) And 0.05 to 0.2 parts by weight of proteolytic enzyme is mixed at a temperature of 40 to 50 DEG C for 2 to 4 hours.

If the content of the protease is less than 0.05 part by weight, the degradation efficiency of collagen is lowered and the production rate of collagen in the form of a low molecular peptide of 3 kDa or less is lowered. When the content of the protease exceeds 0.2 part by weight, The efficiency is not greatly improved, but the manufacturing cost is increased, and the time of the enzyme inactivation step (S107) is prolonged, which is not preferable in terms of productivity.

In addition, the pH set in the collagen decomposition step (S105) is preferably 6.5 to 7.5, and the collagen extracted and filtered collagen is decomposed into proteolytic enzymes through the collagen extraction filtration step (S103) The degraded collagen enzyme degradation product can be obtained. The higher the content of the low molecular peptide contained in the collagen enzyme degradation product, the higher the yield of the collagen low molecular peptide degradation product of 3 kDa or less.

At this time, the protease is mixed with pancreatin and alkaline protease in a weight ratio of 1: 1. Preferably, the pancreatase is derived from a pancreas, and the alkaline protease is Bacillus licheniformis It is preferable to use Foodpro Alkalin Protease (original Protex 6L) derived from Escherichia coli.

The enzyme inactivation step (S107) is a step of inactivating the enzyme contained in the collagen degradation product decomposed through the collagen decomposition step (S105), and the collagen degradation product decomposed through the collagen decomposition step (S105) Lt; 0 > C for 10 to 20 minutes.

The protein enzyme contained in the heated collagen decomposition product at the above temperature and time is inactivated.

The step of filtering (S109) is a step of filtering the collagen degradation product in which the enzyme is inactivated through the enzyme inactivation step (S107). The collagen degradation product in which the enzyme is inactivated through the enzyme inactivation step (S107) It is a step to increase the yield of the low-molecular collagen degradation product in the form of a peptide of 3 kDa or less, which is excellent in skin aging inhibition and wrinkle-reducing effect by filtering with a membrane filter capable of filtering out components exceeding 3 kDa.

Through the above filtration step, a composition for inhibiting skin aging using a donkey shell consisting of a low-molecular collagen degradation product having a peptide form of 3 kDa or less is completed, and a composition for inhibiting skin aging using the donkey skin prepared through the above- The composition is preferably stored in powder form through a lyophilization process.

The composition for inhibiting skin aging using the donkey skin produced through the above process can be applied to most cosmetic formulations such as creams, lotions, essences and toners, which are formulations of general cosmetics. As described above, Cosmetics such as creams, lotions, essences, and toners disclosed in the present invention exhibit anti-aging and anti-wrinkle effects.

Hereinafter, the method for producing a composition for inhibiting skin aging using the disclosed donkey skin and the physical properties of the composition for inhibiting skin aging prepared through the method will be described with reference to examples.

≪ Example 1 >

After washing with hot water flowing through the donkey's skin, it was heated in 100 ° C water for 20 minutes to remove foreign matter (hair and impurities), and the fat layer was removed by a cutting machine on the donkey skin removed from the foreign substance. And cut into a size of 0.5 cm in length and 1 cm in length. The cut donkey shells (100 g) were mixed with 200 ml of water and subjected to primary extraction at 121 ° C and a pressure of 1.5 kgf / cm ² for 1 hour , And the particles having a particle size of 150 micrometers or more were filtered through a filter to prepare a first extract. 100 ml of water was mixed with 100 parts by weight of the filtrate and then with a high-temperature and high-pressure extractor at 121 ° C The mixture was subjected to secondary extraction at a temperature of 1.5 kgf / cm 2 for 1 hour, filtered through a filter capable of filtering at least 150 micrometers, mixed with the primary extract, Water was added to 100 parts by weight of the prepared collagen extract and 0.1 part by weight of proteinase {Pancripsin (product name of pancreatin, Vision BioChem) and Foodpro alkaine protease (former Protax 6L, product name: dupont genencor)}, and enzymatically decomposed at 45 ° C. for 3 hours. The enzyme-degraded collagen degradation product was inactivated at 90 ° C. for 10 minutes for enzyme treatment, and then a low molecular weight 3 kDa or less Enzyme degradation products and 3 kDa excess enzyme degradation products were prepared by freeze - drying the composition for inhibiting skin aging using donkey skin.

The yield, the degree of hydrolysis, and the content of solid content of the composition for inhibiting skin aging using the donkey shell prepared in Example 1 were measured and shown in Table 1 below.

[However, the yield (%) = we used the {hydrolyzate freeze weight after drying (g) / raw material weight (g)} The calculation of × 100, degree of hydrolysis (%) = a {sample in 10% soluble in TCA Protein content / total protein content present in sample} × 100, and the solids content (g / 100 g) was measured using a refractometric sugar meter.

<Table 1>

The yield (%) of the powder below 3 kDa and the yield (%) of the powder over 3 kDa of the composition for inhibiting skin aging using the donkey shell prepared in Example 1 were measured and shown in Table 2 below.

(The calculation of the yield (%) of the powder below 3 kDa and the yield (%) of the powder above 3 kDa = {the lyophilization weight after filtration of the membrane filter (g) / the weight of the raw material (g)

<Table 2>

The ORAC (Oxygen Radical Absorbance Capacity) activity was measured in order to examine the antioxidative activity of the composition for inhibiting skin aging using the donkey skin of 3 kDa or more prepared in Example 1 above. A composition for inhibiting skin senescence (hereinafter referred to as "powder") using a donkey shell of 3 kDa or less extracted from collagen extract in donkey skin and a composition for inhibiting skin senescence using a donkey shell of 3 kDa or more were coated at 0.1, 0.2 and 0.3 mg / ml , And Trolox was used as a standard reagent. Each sample and 25 μl (microliter) of a standard reagent were placed in a black 96-well plate and mixed with 150 μl of 80 nM Fluorescein. The mixture was incubated at 37 ° C. in an incubator And allowed to stand for 15 minutes. After that, 25 μl of 150 mM AAPH (2,2'-Azobis (2-Amidinopropane) Dihydrochloride) was added and mixed well. The mixture was well mixed at 485 nm excitation wavelength at 37 ° C. using a fluorescence microplate reader, The emission wavelength was measured at 520 nm for 60 minutes at 1 minute intervals. Thereafter, the area under the curve (AUC) of the sample and the standard reagent was measured, and a Trolox equivalent of 쨉 m was measured using a regression curve between the standard reagent concentration and the AUC.

As shown in FIG. 2 below, the experiment showed that the powder of 3 kDa or less exhibited higher ORAC activity than the powder of 3 kDa or more at all concentrations.

In addition, ABTS radical scavenging activity was measured to confirm the antioxidative activity of powders below 3 kDa and excess powder. 3 kDa powder and 3 kDa powder were diluted to 0.5, 1 and 5 mg / ml, and Trolox was used as a standard reagent. 10 mL of 7 mM ABTS solution and 10 mL of 2.45 potassium persulphate solution were mixed and subjected to a cow reaction at room temperature for 16 hours to make an ABTS ⁺ radical. The solution in which radicals were generated was diluted at 735 nm such that the absorbance value was 0.700 ± 0.002 to prepare an ABTS ⁺ solution. Thereafter, 50 각각 of each sample was mixed, reacted in a dark room at 30 캜 for 30 minutes, and the absorbance was measured at 735 nm using a spectrophotometer. The ABTS radical scavenging activity was measured by M Trolox equivlent, which shows the antioxidant effect of Trolox, and is shown in the graph of FIG. 3 below.

As shown in FIG. 3, below 3 kDa powder showed higher ABTS radical scavenging activity than 3 kDa powder at all concentrations.

DPPH radical scavenging activity was also measured to confirm antioxidative activity of the powder below 3 kDa and the powder of over 3 kDa. 3 kDa powder and 3 kDa powder were diluted to 5, 10 and 20 mg / ml, and Trolox was used as a standard reagent. 100 μl of each sample and standard reagent was added to each well of a 96-well plate, and 100 μl of DPPH solution was added to each well. The mixture was reacted for 30 minutes at room temperature in a dark room and then subjected to spectrophotometry at 517 nm Absorbance was measured. The DPPH radical scavenging activity was measured using Trolox as a standard and (μM) Trolox equivalent, and the graph of FIG. 4 is shown below.

As shown in FIG. 4, below 3 kDa powder showed DPPH radical scavenging activity higher than 3 kDa powder at all concentrations.

In addition, ferric reducing anti-oxidant power (FRAP) activity was measured to confirm the antioxidative activity of the powder below 3 kDa and the powder of 3 kDa excess. The active ingredient of the powder below 3 kDa and the powder of over 3 kDa was diluted to a concentration of 5, 10 and 20 mg / ml and analyzed. Trolox was used as a standard reagent. The reaction solution contained 10 mM TPTZ (2,4,6-tris (2-pyridyl) -s-triazine) and 20 mM FeCl ₃ .6H ₂ O dissolved in 40 mM acetate buffer (pH 3.6) 1: 1 by weight, and kept at a temperature of 37 캜. After that, 25 μl of each sample and standard reagent and 175 μl of the reaction solution were mixed and allowed to stand in a dark room for 5 minutes, and the absorbance was then quenched at 590 nm. The FRAP activity was measured as (μM) trolox equivalent, which corresponds to the concentration of trolox used as a standard, and is shown in FIG. 5 below.

As shown in FIG. 5 below, the powder of 3 kDa or less showed FRAP radical scavenging activity higher than 3 kDa powder at all concentrations.

In addition, elastase inhibitory activity was measured in order to confirm the skin wrinkle improving effect of the powder of 3 kDa or less. As a sample, a powder of 3 kDa or less fixed at a concentration of 10, 50, 100, 200 or 300 mg / ml was used. 120 μl of 0.2 M Tris-HCl buffer (pH 8), 20 μl of 1.0 mM SANA (N-succinyl- (Ala) 3-p-nitroanilide) and 100 μl of each concentration sample were added and incubated at 25 ° C. for 10 minutes Then, 20 쨉 l of Porcine pancreas elastase enzyme, which had been separated from porcine pancreas at a concentration of 1 U / ml, was added and reacted at 25 캜 for 20 minutes. The absorbance was measured at 405 nm using a spectrophotometer and is shown in FIG. 6 below. At this time, 100 μg / mL of ursolic acid was used as a positive control. In addition, the following formula was used for the inhibition activity of the enzyme.

Elastase inhibition activity (%) = [1- (sample OD-blank OD) / (control OD-blank OD)} 100]

As shown in FIG. 6 below, the experiment showed that the inhibitory activity of the enzyme was high in a concentration-dependent manner, and the powder of less than 3 kDa at a concentration of 300 mg / mL showed the highest inhibitory activity of the enzyme.

In addition, collagenase inhibitory activity was measured in order to confirm the skin wrinkle improving effect of the powder below 3 kDa. As a sample, powders of 3 kDa or less fixed at concentrations of 0.5, 1, 5, 10, 15 and 20 mg / ml were used. 0.1 mL of a 0.2 mg / ml collagenase enzyme was added to 0.25 mL of a 0.3 mg / mL collagenase chromophore substrate, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 6% citric acid 0.5 mL of citric acid was added to stop the enzyme reaction. Then, 1.5 mL of ethylacetate was added, and the supernatant was measured for absorbance at 320 nm and is shown in FIG. 7 below. Collagenase inhibitory activity was calculated using the following formula.

Collagenase inhibition activity (%) = [1 - {sample OD-blank OD) / (control OD-blank OD)} 100]

As shown in FIG. 7 below, the experiment showed that the collagenase inhibitory activity was high in a concentration-dependent manner. Particularly, the powder of 3 kDa or less at a concentration of 20 mg / mL showed the highest collagenase inhibitory activity.

As a result, the powder of 3 kDa or less showed excellent skin aging inhibition effect and skin wrinkle improving effect.

S101; Raw material pretreatment step
S103; Collagen extraction filtration step
S103-1; The first extraction filtration step
S103-2; The second extraction filtration step
S105; Collagen degradation step
S107; Enzyme inactivation step
S109; Filtration step

Claims (6)

A raw material pretreatment step for pretreating the donkey shell;
A collagen extraction filtration step in which the donkey shell pretreated through the raw material pretreatment step is mixed with water and the collagen is extracted by applying a high temperature and a high pressure and then filtered;
A collagen decomposition step of decomposing collagen by mixing the proteolytic enzyme with the collagen extracted and filtered through the collagen extraction filtration step;
An enzyme inactivation step of inactivating the enzyme contained in the collagen degradation product decomposed through the collagen degradation step; And
And a filtration step of filtering the collagen decomposition product in which the enzyme is inactivated through the enzyme inactivation step,
100 parts by weight of the donkey shell pretreated through the raw material pretreatment step is mixed with 150 to 250 parts by weight of water and extracted at a temperature of 110 to 130 ° C and a pressure of 1 to 2 kgf / cm ² for 50 to 70 minutes And a first extraction and filtering step of filtering; And
The first extraction the collagen is extracted donkey shell 100 weight through filtration step parts of water of 80 to 120 parts by weight of the mixture, and the temperature and 1 to a pressure of 2kgf / cm ² of 110 to 130 ℃ 50 to 70 minutes extraction for and filtration And a second extraction and filtration step,
Wherein the enzyme inactivation step comprises heating the collagen degradation product decomposed through the collagen decomposition step at a temperature of 85 to 95 DEG C for 10 to 20 minutes.
delete The method according to claim 1,
Wherein the collagen decomposition step is performed by mixing 0.05 to 0.2 parts by weight of a protease with 100 parts by weight of collagen extracted and filtered through the collagen extraction filtration step at a temperature of 40 to 50 DEG C for 2 to 4 hours. A method for preparing a composition for inhibiting skin aging using the same.
The method according to claim 1 or 3,
Wherein the proteinase is a mixture of pancreatin and alkaline protease in a weight ratio of 1: 1.
delete The method according to claim 1,
Wherein the filtration step comprises a membrane filter capable of filtering out components having a molecular weight exceeding 3 kDa.

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