CN117625717A - Preparation method of hydrangea She Jiaoyuan protein peptide and cosmetic composition - Google Patents
Preparation method of hydrangea She Jiaoyuan protein peptide and cosmetic composition Download PDFInfo
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- CN117625717A CN117625717A CN202310834900.5A CN202310834900A CN117625717A CN 117625717 A CN117625717 A CN 117625717A CN 202310834900 A CN202310834900 A CN 202310834900A CN 117625717 A CN117625717 A CN 117625717A
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- hydrangea
- jiaoyuan
- cosmetic composition
- protein peptide
- collagen
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- DGKDFNDHPXVXHW-UHFFFAOYSA-N hydrangenol Chemical compound C1=CC(O)=CC=C1C1OC(=O)C2=C(O)C=CC=C2C1 DGKDFNDHPXVXHW-UHFFFAOYSA-N 0.000 description 1
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Dermatology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- Birds (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Cosmetics (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Botany (AREA)
- Mycology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a method for preparing a collagen peptide from hydrangea She Jiaoyuan and a cosmetic composition, and more particularly, to a method for preparing a low molecular collagen peptide from hydrangea, and a cosmetic composition for improving wrinkles and moisturizing comprising the low molecular collagen peptide as an active ingredient.
Description
Technical Field
The present invention relates to a cosmetic composition comprising low molecular collagen peptides isolated from hydrangea. More particularly, the present invention relates to a method for separating and processing a low molecular collagen peptide from hydrangea and a cosmetic composition comprising the low molecular collagen peptide as an active ingredient.
Background
Collagen (collagen) is the major component of connective tissue, and is the most abundant fibrous protein in animals including joints, hair, etc., including bones and skin. Collagen is known to have a molecular weight of about 300000Da and is a polypeptide that makes up most tissues of animals.
Collagen, in particular, is a major protein constituting the dermis layer of the skin, and plays an important role in skin elasticity. As the human body ages, the amount of collagen produced naturally decreases and the amount of collagen decomposed increases, and is lost by exposure to external environments such as ultraviolet rays or pressure, resulting in dryness, reduced elasticity, wrinkles, and the like of the skin. Accordingly, collagen is used in various forms such as health functional foods, medicines, cosmetics, etc., and is generally classified into animal collagen and marine collagen.
In recent years, there is a risk that animal collagen extracted from pigskin, cow leather, etc. may transmit foot-and-mouth disease or mad cow disease, etc. to humans, and marine collagen extracted from fish scales, jellyfish, starfish, etc. has serious drawbacks such as heavy metal, fish anaphylaxis, etc. with marine pollution, and thus, there is a growing demand for novel collagen that compensates for these drawbacks.
On the other hand, as the demand for vegetarian foods, pure skin care, etc. increases, interest in plant collagen also increases. Plant collagen is a glycoprotein similar to collagen containing hydroxyproline (hydroxyproline), proline (proline), glycine (glycine) and the like as main constituent amino acids of collagen, and has been attracting attention, not only for improving skin elasticity and aging effects of animal collagen, but also for containing various phytochemicals (phytochemicals) such as polyphenols and flavonoids which cannot be obtained from animal collagen and have excellent antioxidant functions. However, since collagen and peptides are contained only in a very small amount in general plants, it is difficult and uneconomical to use commercially. Although many studies are being conducted on this, satisfactory results have not been obtained yet.
On the other hand, the hydrangea (Hydrangea Macrophylla) is a generic term for plants belonging to the genus hydrangea of the order cornus, and has a characteristic that the flower color varies with the acidity of soil. This is because the color delphinidin (delphinidin) component of the hydrangea ball changes the degree of absorption of aluminum ions depending on the acidity of the soil.
Since ancient times, the flowers, leaves or root causes of the hydrangea have abundant effects of palpitation, relieving fever, strengthening heart and the like. Also, since ancient times, hydrangea leaf has been drunk as tea, and it has been reported that hydrangea leaf has antimalarial effect, immunosuppressive effect, skin whitening effect, etc., and is known as a food which has not only in vivo fat reducing effect but also helps skin moisturization and keeps skin healthy from skin damage caused by ultraviolet rays at the time of ingestion.
Also, cosmetic compositions for preventing and treating neuropsychiatric diseases, relieving inflammation and whitening of the hydrangea are disclosed in korean patent nos. 171962, 1867308 and 14992074. However, such prior documents on hydrangea include only colchicol (hydrangenol), delphinidin, etc. as an active ingredient or include a preparation method based on a simple extract and efficacy, and no report has been made on cosmetic compositions comprising low molecular collagen peptides extracted and processed from hydrangea.
Accordingly, the present invention has been made in an effort to develop a cosmetic composition having excellent skin penetration and absorption rate and high efficacy compared to the existing extracts by developing a separation process for extracting and processing collagen from hydrangea to maximize the processing efficiency of low molecular collagen peptide, thereby preparing and including the low molecular collagen peptide in the cosmetic composition.
Prior art literature
Patent literature
Patent document 1: a method for preparing a tremella enzyme-treated low molecular polysaccharide extract containing plant collagen is disclosed in Korean laid-open patent No. 10-2022-0000746.
Patent document 2: korean patent No. 10-2066435 discloses a method for preparing collagen using olives, olive collagen prepared using the same, and a cosmetic composition comprising the same as an active ingredient.
Patent document 3: a cosmetic composition comprising hibiscus collagen peptide is disclosed in Korean patent No. 10-2299846.
Disclosure of Invention
Technical problem
The invention aims to provide a method for extracting collagen peptide from hydrangea and processing the collagen peptide into low molecular peptide. And, it is intended to develop and provide a cosmetic composition having excellent skin penetration rate and absorption rate and exhibiting high functionality by using it as a raw material.
Solution to the problem
The invention provides a preparation method of hydrangea She Jiaoyuan protein peptide, which comprises the following steps: step (a) of inducing a first enzymatic reaction by treating hydrangea leaves with bacillus-derived subtilisin (subtilisin); and a step (b) of inducing a second enzymatic reaction by treating pepsin after the step (a).
In the present invention, preferably, the preparation method of the hydrangea She Jiaoyuan protein peptide may further include: step (c), after the step (b), recovering the first supernatant by centrifugation, adding ethanol and standing at a low temperature; step (d) of recovering a second supernatant excluding the precipitate by centrifugation after the step (c); and (e) recovering a filtrate by filtering the recovered second supernatant after the step (d).
In addition, in the present invention, more preferably, the method for preparing the hydrangea She Jiaoyuan protein peptide may further include: and (f) after the step (e), dissolving or suspending the recovered filtrate or the lyophilized powder obtained by freeze-drying the filtrate in distilled water, and then obtaining a low molecular peptide having a molecular weight of 300Da or less by gel chromatography.
In the present invention, it is preferable that the first enzymatic reaction of the above step (a) is performed at a pH of 6 to 8 and the second enzymatic reaction of the above step (b) is performed at a pH of 2 to 4.
In the present invention, it is preferable that the first enzymatic reaction in the step (a) is carried out at a temperature of 50℃to 60℃under a pressure of 20 to 100bar, and the second enzymatic reaction in the step (b) is carried out at a temperature of 35℃to 45℃under a pressure of 20 to 100 bar.
The present invention provides a cosmetic composition comprising the sparassis crispa She Jiaoyuan protein peptide prepared by the above method of the present invention.
In the cosmetic composition of the present invention, preferably, the above cosmetic composition may be used for skin moisturization or for improving skin wrinkles.
In the cosmetic composition of the present invention, as an example, the cosmetic composition may have any one of dosage forms selected from the group consisting of a lotion, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water (O/W) cosmetic, a water-in-oil (W/O) cosmetic, an ointment, a foundation, a concealer, a lipstick, and a scalp cosmetic.
ADVANTAGEOUS EFFECTS OF INVENTION
The present invention provides a method for preparing a low molecular collagen peptide from a plant collagen peptide prepared from hydrangea and separating it according to molecular weight, and a cosmetic composition comprising the same. Also, the cosmetic composition of the present invention has a matrix metalloproteinase-1 (MMP-1) production inhibiting effect, a wrinkle preventing effect by increasing the collagen synthesis amount, and a moisturizing effect by increasing aquaporin 3 (AQP-3) expression.
Drawings
FIG. 1 shows the results of confirming the molecular weight of the hydrangea She Jiaoyuan protein peptide and the hydrangea low molecular collagen peptide.
FIG. 2 shows the results of analysis of amino acids of low molecular collagen peptides of hydrangea leaf.
FIG. 3 shows the results of measurement of the MMP-1 production inhibitory effect of the low molecular collagen peptide of hydrangea leaf.
FIG. 4 shows the results of measuring the efficacy of increasing AQP-3 expression of low molecular collagen peptide from hydrangea leaf.
FIG. 5 shows the results of measuring the collagen synthesis increasing effect of the low molecular weight collagen peptide of hydrangea leaf.
Detailed Description
The invention provides a method for preparing collagen peptide from hydrangea leaf. The preparation method of the hydrangea collagen peptide is carried out by the following steps.
< step (a): first enzyme reaction ]
This step is a process of inducing a first enzymatic reaction by treating hydrangea leaf with bacillus-derived subtilisin.
Preferably, the hydrangea leaves used in this step may be crushed or broken using a pulverizer or breaker. Also, it is preferable that the hydrangea leaf is soaked in purified water for use to perform an enzyme reaction. In this case, the first enzyme reaction is preferably performed at a pH of 6 to 8, and the optimal enzyme reaction is preferably induced by adjusting the pH of the first enzyme reaction solution to a pH of 6 to 8.
Preferably, the first enzymatic reaction of this step may be carried out using an ultra-high pressure enzyme reactor containing a polymer coated with an enzyme of Bacillus subtilis origin, preferably maintained at a temperature of 50℃to 60℃under a pressure of 20 to 100 bar. In this case, the reaction time is preferably about 12 to 36 hours.
< step (b): second enzymatic reaction ]
This step is a process of inducing a second enzymatic reaction by treating pepsin after the above step (a).
After the first enzymatic reaction, a second enzymatic reaction is induced by post-treating pepsin after recovering the reacted solution. In this case, the second enzyme reaction is preferably carried out at a pH of 2 to 4. The optimal enzyme reaction is induced by adjusting the pH of the second enzyme reaction solution to 2 to 4.
Preferably, the second enzymatic reaction of this step can be carried out in an ultra-high pressure enzymatic reactor, which is filled with pepsin-coated polymer, preferably maintained at a temperature of 35-45 ℃ at a pressure of 20-100 bar. In this case, the reaction time is preferably 3 to 9 hours.
Preferably, after the second enzymatic reaction of the present step is completed, the remaining enzymes are inactivated by boiling the solution after the second reaction.
< step (c): recovering the first supernatant and ethanol precipitation ]
This step is a process of recovering the first supernatant by centrifugation after the above step (b), adding ethanol and leaving at a low temperature.
In this step, the first supernatant is recovered by centrifuging the solution after the second reaction of step (c) above and ethanol is added to induce ethanol precipitation. Ethanol can be added to the supernatant at this time in a weight ratio of 1:1. When ethanol is added in this way, precipitation is induced to sink the precipitate to the bottom. Preferably, the ethanol precipitation is performed at refrigeration temperatures.
< step (d); recovery of the second supernatant ]
This step is a process of recovering the second supernatant excluding the precipitate by centrifugation after the above step (c). The second supernatant from which the precipitate was removed can be recovered by this process.
< step (e): recovering filtrate)
This step is a process of recovering a filtrate by filtering the recovered second supernatant after the step (d). Preferably, filtration is performed using a filtration membrane of 0.1 to 0.5 μm. After filtration, the filtrate may be freeze-dried as the case may be.
< step (f): gel chromatography ]
The step is a process in which the recovered filtrate or lyophilized powder obtained by freeze-drying the filtrate is dissolved or suspended in distilled water after the step (e), and then a low-molecular peptide having a molecular weight of 300Da or less is obtained by gel chromatography.
The low molecular peptide can be obtained by this step, and the chromatography can be performed by "size exclusion chromatography".
After the gel chromatography in this step, the resulting solution may be freeze-dried as the case may be.
In another aspect, the invention provides a cosmetic composition comprising the hydrangea She Jiaoyuan protein peptide prepared by the above method of the invention.
It was confirmed that the cosmetic composition comprising the low molecular collagen peptide derived from hydrangea as described above is effective in improving wrinkles and moisturizing. The cosmetic composition comprising a low molecular collagen peptide of the present invention is rich in glycine, proline, hydroxyproline, etc. as the main amino acid sequence of collagen, and has a molecular weight of 300Da or less to improve the biological absorbability, thereby providing an improved wrinkle-improving effect and an enhanced moisturizing effect as compared with the conventional extracts and collagen.
In the cosmetic composition of the present invention, as an example, the cosmetic composition may have any one of dosage forms selected from the group consisting of a lotion, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water type cosmetic, a water-in-oil type cosmetic, an ointment, a foundation, a concealer, a lipstick, and a scalp cosmetic.
Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the scope of the invention required by the present invention is not limited to the following examples and experimental examples, and includes modifications of technical ideas equivalent thereto.
Example 1: preparation of Sparassis crispa She Jiaoyuan protein peptide
The above-mentioned hydrangea She Jiaoyuan protein peptide was prepared by the following method.
(1) Powdering hydrangea leaf: by pulverizing hydrangea She Dedao hydrangea leaf powder.
(2) Enzyme decomposition of hydrangea leaf: soaking the hydrangea leaf powder in purified water, regulating the pH of the solution to 6-8, and then placing the hydrangea leaf powder in an ultrahigh pressure enzyme reactor containing a polymer coated with bacillus-derived subtilisin, and maintaining the temperature of 50-60 ℃ for 24 hours under the pressure of 20-100 bar to perform enzymatic decomposition reaction. After recovering the solution from the reactor, the pH of the solution is adjusted to 2 to 4, and then the solution is placed in an ultra-high pressure enzyme reactor containing a polymer coated with pepsin, and an enzymatic decomposition reaction is performed for 6 hours while maintaining a temperature of 35 to 45 ℃ at a pressure of 20 to 100bar, and then the remaining enzyme is deactivated by boiling for 30 minutes to terminate the reaction, and then the solution is recovered and the supernatant is obtained by centrifugation.
(3) Separating and purifying: ethanol was added to the supernatant at a weight ratio of 1:1 and left at a low temperature, and then a filtrate was obtained by centrifuging the supernatant excluding the precipitate and passing through a microfiltration membrane of 0.1 to 0.5 μm.
(4) And (3) drying: the hydrangea She Jiaoyuan protein peptide powder is obtained by concentrating the filtrate under reduced pressure at a temperature of 50-60 ℃ and freeze-drying.
Example 2: preparation of hydrangea leaf low molecular collagen peptide
The hydrangea leaf low molecular collagen peptide of this example 2 was prepared by the following method.
(1) Filling gel chromatography resin and activating: gel chromatography resin Sephadex LH-20 (Sigma-Aldrich, USA) was loaded into an open column after Swelling (Swelling) with 1-2 times the volume ratio of "distilled water subjected to the degassing (de-gas) process". Distilled water 2 to 3 times the resin volume was then passed through to stabilize it.
(2) Separating and purifying: the dried hydrangea She Jiaoyuan protein peptide powder of example 1 above was dissolved in distilled water and loaded in a specified amount (Loading) into a open tubular column. Then, distilled water 5 times the resin capacity was continuously flowed at an appropriate speed to wash the substances having a large molecular weight. Then, distilled water, which is 5 to 10 times the resin capacity, is continuously flowed at an appropriate speed to obtain an eluent.
(3) And (3) drying: concentrating the eluent under reduced pressure at 50-60 ℃ and freeze-drying to obtain the hydrangea leaf low molecular collagen peptide powder with molecular weight below 300 Da.
Comparative example 1: preparation of hydrangea leaf extract
The hydrangea leaf extract of comparative example 1 was prepared by the following method.
(1) Powdering hydrangea leaf: by pulverizing hydrangea She Dedao hydrangea leaf powder.
(2) Extracting hydrangea leaf: soaking the obtained hydrangea leaf powder in purified water of 5-10 times, and extracting at 50-60deg.C for 24 hr.
(3) And (3) drying: the filtrate is obtained by centrifugation and passing through a microfiltration membrane of 0.1-0.5 μm, and the filtrate is concentrated under reduced pressure at a temperature of 50-60 ℃ and freeze-dried to obtain hydrangea leaf extract powder.
Comparative example 2: preparation of Sparassis crispa She Jiaoyuan protein by the existing method
The hydrangea She Jiaoyuan protein was prepared by the following prior methods. The existing hydrangea She Jiaoyuan protein was prepared by the method disclosed in the existing literature or patent using the method of korean patent No. 10-2299846.
(1) Powdering hydrangea leaf: by pulverizing hydrangea She Dedao hydrangea leaf powder.
(2) Extraction and enzyme treatment: to the resulting hydrangea leaf powder, 1 to 5 times of water was added and heat-treated at 80℃for 60 minutes, then the pH was adjusted to 4.0, and trypsin was then added and hydrolyzed at 50℃for 10 hours. It was heated at a temperature of 85℃for 10 minutes to inactivate the remaining enzymes to terminate the reaction.
(3) And (3) drying: the filtrate is obtained by centrifugation and passing through a microfiltration membrane of 0.1-0.5 μm, and the filtrate is concentrated under reduced pressure at a temperature of 50-60 ℃ and freeze-dried to obtain hydrangea She Jiaoyuan protein powder.
Experimental example 1: analysis of protein content
The Protein content of examples 1-2 and comparative examples 1-2 prepared above was determined using a bicinchoninic acid (Bicinchoninic acid, BCA) Protein Assay (Protein Assay).
BCA protein assay reagents (BCA Protein Assay Reagent) a and B (Thermo Fisher Scientific, usa) were mixed in a 50:1 ratio and 175 μl each was dispensed in a 96 well plate, then 25 μl of the diluted 0.1% strength sample was treated and reacted in an incubator at 37 ℃ for 30 minutes, and absorbance was measured at 540 nm. Protein content was calculated after absorbance was measured using bovine serum albumin (Bovine serum albumin, BSA) as a standard substance, and the results are shown in table 1.
TABLE 1
From table 1, it was confirmed that the protein contents of examples 1 and 2 were more excellent than those of comparative examples 1 and 2. This means that the protein extraction efficiency by the present invention is more excellent than the existing extracts and collagen extraction methods.
Experimental example 2: determination of molecular weight
The molecular weights of examples 1-2 prepared above were determined using a Matrix assisted laser desorption ionization time of flight mass spectrometer (MALDI-TOF MS, matrix-assisted laser desorption-ionization time-of-flight mass spectrometer) and a gas chromatograph/mass spectrometer (GC/MS, gas chromatography/mass spectrometer). Analysis was performed with respect to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer using a Microflex LRF MALDI-TOF mass spectrometer and Bruker UltrafleXtreme (Bruker Daltonics, japanese apricot City (Bremen), germany), and with respect to a gas chromatograph/mass spectrometer using an Agilent 8890/5977B GC/MSD (Agilent Technologies, california, U.S. A. (USA)). The results are shown in fig. 1.
It is understood that the molecular weight distribution of example 1 was 1000Da or less, the molecular weight distribution of example 2 was 300Da or less, and most of the molecular weight was distributed. This means that the above-mentioned example 2 well separated the low molecular collagen peptide from example 1 by using a gel chromatography resin.
Experimental example 3: analysis of amino acids
The amino acid was analyzed using an amino acid analyzer to analyze the amino acid composition of example 2 prepared as described above. The results were analyzed by an L-8900amino acid analyzer (L-8900 Amino Acid Analyzer, hitachi, tokyo, japan) and are shown in FIG. 2 and Table 2.
TABLE 2
From FIG. 2 and Table 2 above, it was confirmed that the amino acid composition of example 2 was rich in glycine, proline, hydroxyproline, etc. as the main amino acid sequence of collagen.
Experimental example 4: determination of MMP-1 production inhibitory Effect
The MMP-1 production inhibitory effect of examples 1 and 2 and comparative examples 1 and 2 prepared as described above was confirmed. Human skin fibroblasts (HDFn) were cultured at 5X 10 4 Individual cells/wells were dispensed into 24-well plates and then incubated at 37℃with 5% CO 2 1J/cm post-treatment after 1 day stabilization in a cell incubator 2 Long wave Ultraviolet (UVA) radiation. Each sample was diluted and treated at different concentrations while changing to a medium supplemented with 2% Fetal Bovine Serum (FBS), and Adenosine (wrinkle-functional bulletin raw material) was treated at 400 μm concentration and reacted for 48 hours as a positive control group, and then R was used&The MMP-1 expression level of the supernatant was confirmed by the D systems kit (kit) (DY 901), and cytotoxicity was confirmed by treating the pellet with MTT Solution (MTT Solution).
After MMP-1 enzyme-linked immunosorbent Assay (MMP-1 ELISA Assay), the amount of generated MMP-1 was calculated by measuring absorbance at a wavelength of 450nm, and then the MMP-1 expression inhibition (%) was calculated as compared with the control group, the calculation formula of which is shown in the following equation 1.
Mathematical formula 1:
the experimental results are shown in fig. 3. As shown in FIG. 3, it was confirmed that MMP-1 production inhibitory activity was higher in treatment example 2 than in treatment example 1 or comparative examples 1 to 2. That is, it was confirmed that although example 1 and comparative examples 1 and 2 also effectively inhibited the production of MMP-1, the MMP-1 production inhibitory activity of example 2 consisting of the low molecular collagen peptide isolated from the hydrangea leaf was higher.
Experimental example 5: determination of AQP-3 expression increasing Effect
The AQP-3 expression increasing effect of examples 1 to 2 and comparative examples 1 to 2 prepared above was confirmed. Human skin keratinocytes (HaCaT) were cultured at 3X 10 5 Individual cells/wells were dispensed into 6-well plates and then incubated at 37℃with 5% CO 2 After stabilization in the cell incubator for 1 day, each sample was diluted at different concentrations and treated, and Dexamethasone (Dexamethasone) was treated and reacted for 6 hours, and then cells were collected using a scraper as a positive control group. After isolating the proteins using a cell lysis buffer and quantifying the proteins by BCA assay (BCA assay), the same amount of proteins were electrophoresed in a 10% SDS-PAGE gel. The proteins separated on the gel were transferred to PVDF membrane and blocked with 5% skim milk for 1 hour. After washing with TTBS solution, primary antibody (AQP-3, santa cruz, usa) was reacted at a temperature of 4 ℃ for 1 day, and then HRP-conjugated secondary antibody was reacted at room temperature for 2 hours. After washing with TTBS solution, ECL solution was treated and bands were detected with Chemi Doc instrument, and AQP-3 expression was calculated based on beta actin expression and confirmed using Image J1.47 software (software).
The experimental results are shown in fig. 4 below. As shown in FIG. 4, it was confirmed that AQP-3 expression was highly increased in treatment example 2 compared with treatment example 1 or comparative examples 1 to 2. That is, although example 1 and comparative examples 1 and 2 also effectively increased the expression of AQP-3, the AQP-3 expression-increasing activity of example 2 consisting of the low molecular collagen peptide isolated from hydrangea leaf was higher, and thus excellent moisturizing effect could be expected.
Experimental example 6: measurement of collagen production-promoting Effect
The collagen production promoting effect of examples 1 to 2 and comparative examples 1 to 2 prepared as described above was confirmed. Human skin fibroblasts (HDFn) were cultured at 1X 10 5 Individual cells/wells were dispensed into 24-well plates and then incubated at 37℃with 5% CO 2 After stabilizing for 1 day, 500 μm cortisone and each sample diluted at various concentrations, and TGF- β was treated and reacted for 24 hours as a positive control group, then the collagen expression level of the supernatant was confirmed by using type i Procollagen peptide EIA kit (protocol type 1peptide EIA kit), and cytotoxicity was confirmed by treating the precipitate with MTT Solution (MTT Solution). After the collagen enzyme-linked immunosorbent assay (Collagen ELISA Assay), the amount of collagen produced was calculated by measuring absorbance at a wavelength of 450nm, and then the increase rate (%) of collagen expression compared to the control group was calculated.
The experimental results are shown in fig. 5 below. As shown in fig. 5, it was confirmed that the collagen expression increasing activity was higher in the treatment example 2 than in the treatment example 1 or the comparative examples 1 to 2. That is, although example 1 and comparative examples 1 and 2 also effectively increased collagen expression, example 2, which consisted of low molecular collagen peptides isolated from hydrangea leaves, had higher collagen expression increasing activity, and thus, excellent wrinkle-improving effects could be expected.
Dosage form examples 1 to 5: preparation of cosmetic composition dosage forms
Cosmetic compositions of dosage form examples 1 to 5 having compositions shown in the following table 3 were prepared. Formulation examples 2 to 5 are cosmetic compositions of comparative examples 1 to 2 and examples 1 to 2, respectively, added to the cosmetic composition prepared by formulation example 1.
TABLE 3 Table 3
Experimental example 7: measuring wrinkle-improving Effect
In order to conduct a human body application test on the skin wrinkle improvement effect of the above-prepared dosage forms 1 to 5, the wrinkle improvement effect before and after 10 weeks of use of the dosage forms 1 to 5 was evaluated by measuring by an instrument (cutometer SEM 575,C+K Electronic, germany) on 25 women aged 20 to 40 who need to improve skin. The experiment was performed in such a way that group a applied formulation example 1 to the face, group B applied formulation example 2 to the face, group C applied formulation example 3 to the face, group D applied formulation example 4 to the face, and group E applied formulation example 5 to the face.
TABLE 4 Table 4
The experimental results are shown in table 4 above. As shown in table 4, it was confirmed that the wrinkle-improving effect was excellent in the treatment dosage form example 5 as compared with the treatment dosage form examples 1 to 4. It was confirmed that the embroidered ball leaf low molecular collagen peptide of example 2 had a higher wrinkle-improving effect, although the embroidered ball She Jiaoyuan protein peptide of example 1 and the comparative examples 1 and 2, which were conventional embroidered ball leaf extracts and the embroidered ball She Jiaoyuan proteins, had wrinkle-improving effects.
Experimental example 8: determination of skin moisture content
In order to measure the skin moisturizing effect of the formulation examples 1 to 5 prepared above, the skin surface moisture content before and after the use of the formulation examples 1 to 5 was measured by a skin moisture tester (corneometer). The skin moisture tester for measuring moisture retention is a device for measuring moisture content of skin surface by capacitance measurement (capacitance measurement) under constant temperature and humidity conditions in which room temperature of 20 to 25 ℃ and relative humidity of 40 to 55% are maintained, and the measurement unit is expressed as arbitrary unit (a.u., arbitrary unit) given by the device. The initial values before the application of the test samples were determined, and then the skin moisture content was determined at various times using a skin moisture tester.
TABLE 5
| A.U. | 0 min (min) | For 10 minutes | 30 minutes | 60 minutes | 90 minutes | 180 minutes |
| Formulation example 1 | 31.6 | 40.5 | 39.3 | 36.1 | 33.7 | 31.1 |
| Formulation example 2 | 30.7 | 42.9 | 40.2 | 37.4 | 35.1 | 31.5 |
| Formulation example 3 | 31.8 | 43.1 | 42.5 | 38.4 | 35.5 | 32.7 |
| Formulation example 4 | 31.5 | 44.2 | 43.1 | 38.6 | 35.8 | 32.4 |
| Formulation example 5 | 30.4 | 45.4 | 44.5 | 41.8 | 38.4 | 35.1 |
The experimental results are shown in table 5 above. As shown in table 5, it was confirmed that the skin moisture content was significantly increased in treatment formulation example 5 compared with treatment formulation examples 1 to 4. This is because, although the conventional hydrangea leaf extract and hydrangea She Jiaoyuan proteins of comparative examples 1 to 2 and the hydrangea She Jiaoyuan protein peptide of example 1 also increase the moisture content of the skin, the hydrangea leaf low-molecular collagen peptide of example 2 increases the moisture content of the skin more significantly, and thus a more excellent moisturizing effect can be expected.
Claims (8)
1. A method for preparing a hydrangea She Jiaoyuan protein peptide, comprising the steps of:
step (a) of inducing a first enzymatic reaction by treating hydrangea leaves with bacillus-derived subtilisin; and
step (b) of inducing a second enzymatic reaction by treating pepsin after step (a) above.
2. The method for preparing the hydrangea She Jiaoyuan protein peptide according to claim 1, wherein the method for preparing the hydrangea She Jiaoyuan protein peptide further comprises:
step (c), after the step (b), recovering the first supernatant by centrifugation, adding ethanol and standing at a low temperature;
step (d) of recovering a second supernatant excluding the precipitate by centrifugation after the step (c); and
and (e) recovering a filtrate by filtering the recovered second supernatant after the step (d).
3. The method for preparing the hydrangea She Jiaoyuan protein peptide according to claim 2, wherein the method for preparing the hydrangea She Jiaoyuan protein peptide further comprises:
and (f) after the step (e), dissolving or suspending the recovered filtrate or the lyophilized powder obtained by freeze-drying the filtrate in distilled water, and then obtaining a low molecular peptide having a molecular weight of 300Da or less by gel chromatography.
4. The method of producing a protein peptide of hydrangea She Jiaoyuan according to claim 1, wherein the first enzymatic reaction of step (a) is carried out at a pH of 6 to 8 and the second enzymatic reaction of step (b) is carried out at a pH of 2 to 4.
5. The method of producing a protein peptide of hydrangea She Jiaoyuan according to claim 1, wherein the first enzymatic reaction of step (a) is carried out at a temperature of 50 ℃ to 60 ℃ under a pressure of 20 to 100bar, and the second enzymatic reaction of step (b) is carried out at a temperature of 35 ℃ to 45 ℃ under a pressure of 20 to 100 bar.
6. A cosmetic composition comprising the hydrangea She Jiaoyuan protein peptide prepared by the method of any one of claims 1 to 3.
7. The cosmetic composition according to claim 6, wherein the cosmetic composition is used for skin moisturization or for improving skin wrinkles.
8. The cosmetic composition according to claim 6, wherein the cosmetic composition has any one of dosage forms selected from the group consisting of a lotion, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water type cosmetic, a water-in-oil type cosmetic, an ointment, a foundation, a concealer, a lipstick, and a scalp cosmetic.
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