KR102657397B1 - Multifunctional cosmetic composition containing multi-molecular collagen and Kakadu plum extract - Google Patents

Abstract

After studying natural product extracts that can double and diversify the effects when administered in combination with low-molecular-weight collagen, the present invention found that using Kakadu plum extract improves skin moisturization, skin whitening, skin elasticity, and The present invention has been completed by confirming that the cosmetic composition according to the present invention has a significantly improved effect on skin improvement, skin wrinkle prevention, etc., and the cosmetic composition according to the present invention includes multimolecular collagen having a low molecular weight and various molecular weight distribution; and Kakadu plum fruit extract as an active ingredient, and is characterized by useful effects in skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles.

Description

Multifunctional cosmetic composition containing multi-molecular collagen and Kakadu plum extract as active ingredients {Multifunctional cosmetic composition containing multi-molecular collagen and Kakadu plum extract}

The present invention relates to a cosmetic composition containing multimolecular collagen and Kakadu plum extract. More specifically, it contains multimolecular collagen and Kakadu plum extract as active ingredients to moisturize the skin, whiten the skin, maintain and improve skin elasticity, It relates to a multi-functional cosmetic composition that is effective in preventing skin wrinkles, etc.

Various changes occur in the skin as it ages. First, the thickness of the epidermis, dermis, and subcutaneous tissue, which are the components of the skin, becomes thinner, and the extracellular matrix (ECM), which gives skin elasticity, changes. Collagen, which accounts for 70-80% of the matrix, rapidly declines in production with age and is closely related to the formation of wrinkles. Collagen, elastin, and glycosaminoglycan, which make up skin connective tissue, (glucosaminoglycan), fibronectin, etc. are oxidized and lose their functions, causing the skin to lose elasticity and excessive wrinkles to form, turning it into senile skin.

Connective tissue fibers of the extracellular matrix include glue fibers (collagen fibers), reticular fibers, and elastic fibers (elastic fibers), of which collagen accounts for about 70% of skin connective tissue. It is mostly formed in fibroblasts of the skin.

As we age, the collagen content in skin connective tissue decreases, which is due to a decrease in collagen synthesis and acceleration of decomposition. Therefore, a decrease in collagen biosynthesis and acceleration of collagen decomposition can be said to be the biggest cause of skin wrinkle formation. The collagen biosynthesis process is controlled and changed by many factors involved at the transcription level and post-translation level, and collagen decomposition is caused by collagen decomposition by ultraviolet rays, etc. By promoting the expression of matrix metalloproteases (MMP) such as collagenase, collagen decomposition is promoted and collagen content is reduced. In addition, as the transformation of collagen is accelerated by the external environment, wrinkles increase and deepen on the skin. Under this technical background, research is being actively conducted to utilize the skin moisturizing effect and wrinkle-improving effect of conventional collagen, elastin, etc. (Korea Registered Patent No. 10-1700903). However, the transdermal absorption of high molecular substances such as collagen, elastin, etc. There is a problem that it is difficult.

In order to solve this problem, research on cosmetic compositions for preventing wrinkles and maintaining skin elasticity using low-molecular-weight collagen that is easily percutaneously absorbed (Korean Patent Publication No. 10-2022-0164865 and Korean Patent Publication No. 10-2021 -0127844) is in progress, but there are limits to achieving the goal with simple low-molecular-weight collagen alone, so a new combination of cosmetic compositions is needed.

1. Republic of Korea Patent No. 10-1700903 2. Republic of Korea Patent Publication No. 10-2022-0164865 3. Republic of Korea Patent Publication No. 10-2021-0127844

Accordingly, the present inventor studied natural product extracts that can double and diversify the effects when administered in combination with low-molecular-weight collagen. As a result, the use of Kakadu plum extract improved skin moisturization, skin whitening, maintenance of skin elasticity, and The present invention has been completed by confirming that it has a significantly improved effect on skin improvement, skin wrinkle prevention, etc., and the problem to be solved by the present invention is to moisturize the skin by containing multimolecular collagen and Kakadu plum extract, which are low molecular and have various molecular weight distributions, as active ingredients. , to provide a multifunctional cosmetic composition that is effective for skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles.

In order to achieve the above technical problem, the present invention

Multimolecular collagen with low molecular weight and diverse molecular weight distribution; and

Provided is a composition that contains Kakadu plum fruit extract as an active ingredient and is effective in moisturizing the skin, whitening the skin, maintaining and improving skin elasticity, and preventing skin wrinkles.

In the multifunctional cosmetic composition according to the present invention as described above, the multimolecular collagen may have a molecular weight distribution of 200 to 5,000 Da.

In the multifunctional cosmetic composition according to the present invention as described above, the Kakadu plum fruit extract may be an extract using water or ethanol as an extraction solvent.

As described above, the multifunctional cosmetic composition according to the present invention is characterized in that it consists of 95 to 99 parts by weight of the multimolecular collagen and 1 to 5 parts by weight of the Kakadu plum fruit extract based on 100 parts by weight of the total cosmetic composition.

The composition of the present invention adds a small amount of Kakadu plum extract to multimolecular collagen with a low molecular weight and diverse molecular weight distribution, and has clinically effective effects such as moisturizing the skin, whitening the skin, maintaining and improving skin elasticity, and preventing skin wrinkles, making it a cosmetic raw material. As such, its value is very high.

Hereinafter, specific details for easily carrying out the present invention will be described in detail through examples and the like.

After researching natural product extracts that can double and diversify the effects when administered in combination with low-molecular-weight collagen, the present invention found that using Kakadu plum extract moisturizes the skin, whitens the skin, and maintains and improves skin elasticity compared to when only low-molecular-weight collagen is used. , the present invention was completed by confirming that it showed a significantly improved effect in preventing skin wrinkles, etc.

The cosmetic composition according to the present invention is

Multimolecular collagen with low molecular weight and diverse molecular weight distribution; and

It contains Kakadu plum fruit extract as an active ingredient and is characterized by useful effects in skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles.

In the composition according to the present invention as described above, the multimolecular collagen is not particularly limited as long as the manufacturing source of the collagen is widely known in the technical field to which the present invention belongs, such as animal or vegetable, and can be used as long as it has a molecular weight distribution that facilitates transdermal absorption. and preferably has a molecular weight distribution of 200 to 5,000 Da.

In addition, the Kakadu plum (Terminalia Ferdinandiana) of the present invention is a tree (Terminalia ferdinandiana) of the Combretaceae family of Myrtales. The tree is thin and 32 m tall, and the fruit is yellowish green and has a length of 2. cm, and the diameter is about 1 cm. The fruit contains a fairly large amount of vitamin C, 3.2 to 5.0 g per 100 g, and the fruit is used in the present invention.

The extraction method from the cacao plum fruit extract as described above can be used without particular limitation as long as it is widely known in the technical field to which the present invention pertains. Examples include water, lower alcohols with 1 to 4 carbon atoms, glycerin, ethylene glycol, and propylene glycol. And butylene glycol is extracted with at least one solvent selected from the group consisting of, filtered, and then concentrated under reduced pressure to prepare a composite extract. Preferably, water or ethanol is used as the extraction solvent, and more preferably, water is used as the extraction solvent.

In the multifunctional cosmetic composition according to the present invention as described above, the weight ratio of the multimolecular collagen and the Kakadu plum fruit extract is not particularly limited, but the total composition consisting of the multimolecular collagen and the Kakadu plum fruit extract is 100. It is preferably composed of 95 to 99 parts by weight of the multimolecular collagen and 1 to 5 parts by weight of the kakadu plum fruit extract. Even if only 1 to 5 parts by weight of the kakadu plum fruit extract is included, it is more effective than when containing only low molecular collagen. This is based on confirmed results showing significantly improved effects in skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles.

Additionally, the composition of the present invention may further contain known substances that exhibit cosmetic effects. Specifically, it may further contain one or more selected from the group consisting of ascorbyl glucoside, licorice extract, arbutin, ascorbic acid, kojic acid, and niacinamide.

The composition of the present invention provides excellent antioxidant power, prevents skin aging through skin whitening, increases skin elasticity or improves skin wrinkles, improves skin moisturizing ability, improves skin troubles such as acne, sebum control effect, and pore shrinkage effect. , it can provide an overall improvement effect on the overall skin condition, such as an effect of improving keratin, skin converging or refining effect, improving complexion by improving blood circulation, alleviating skin irritation, and improving homeostasis.

The composition according to the present invention may be formulated containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example, solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranulocytes or ionic forms (liposomes) and non-ionic forms. It may be provided in the form of an ionic vesicular dispersion, or in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. It can also be used in the form of foam or in the form of an aerosol composition further containing compressed propellant. These compositions can be prepared according to conventional methods in the art.

Additionally, the composition according to the present invention may contain fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic. Ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetics. It may contain adjuvants commonly used in the fields such as cosmetology or dermatology. The auxiliaries are introduced in amounts commonly used in the fields of cosmetology or dermatology.

Additionally, the composition of the present invention may contain a substance that promotes skin absorption to increase the skin beauty effect.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples and test examples. However, these examples and test examples are provided only for illustrative purposes to aid understanding of the present invention, and the scope and scope of the present invention are not limited by the examples below.

<Example and comparative example composition preparation>

1. Example 1

A composition according to the present invention was prepared to include fish collagen with a molecular weight distribution of 200 to 5,000 Da and ethanol extract of Kakadu plum fruit at a weight ratio of 95:1.

The Kakadu plum fruit ethanol extract is obtained by adding 5 times the weight of 50% ethanol aqueous solution to Kakadu plum fruit powder (200mesh), extracting it at 50°C for 5 hours, filtering, concentrating under reduced pressure, and freeze-drying to obtain the Kakadu plum fruit ethanol extract. was manufactured.

2. Example 2

It is the same as Example 1, except that the weight ratio is 95:5.

3. Example 3

It is the same as Example 1, but the difference is that hot water extract was used as the Kakadu plum fruit extract.

The Kakadu plum fruit hot water extract was extracted by adding 5 times the volume of water to Kakadu plum fruit powder (200mesh) at about 100°C for 60 minutes. The obtained extract was separated and removed using a centrifuge at 8,000 rpm for 20 minutes, then concentrated under reduced pressure and freeze-dried to prepare a hot water extract of Kakadu plum fruit.

4. Example 4

It is the same as Example 2, but the difference is that hot water extract was used as the Kakadu plum fruit extract.

5. Comparative Example 1

A cosmetic composition was prepared using only fish collagen with a molecular weight distribution of 200 to 5,000 Da.

6. Comparative Example 2

A cosmetic composition was prepared using only Kakadu plum fruit thermal water extract.

7. Comparative Example 3

A cosmetic composition was prepared using only the ethanol extract of Kakadu plum fruit.

<Test Example 1: In-vitro whitening efficacy evaluation>

1) Measurement of tyrosinase inhibitory activity

● Experimental method

Add 0.5 ml of 0.175M sodium phosphate buffer (pH 6.8), 0.2 ml of 10mM L-DOPA as a substrate solution, and 0.1 ml of sample solution into a 96 well plate and mix. Add 0.2 ml of mushroom tyrosinase (110 U/ml) to the mixed solution, react at 25°C for 2 minutes, and measure the DOPA chrome produced in the reaction solution at 475 nm. Tyrosinase inhibitory activity is expressed by the rate of decrease in absorbance of the sample solution added and not added.

Inhibition rate (%) = 1-(Absorbance of sample-added group/Absorbance of sample-free group) x 100

● Experiment results

Tyrosinase plays a very important role in the production of skin melanin. It acts as a tyrosine hydroxylase, which oxidizes tyrosine within the melanosome to create DOPA, and as a DOPA oxidase, which oxidizes DOPA to create DOPA quinone, acting as an important enzyme in synthesizing melanin polymer. The whitening effect can be determined through tyrosinase inhibitory activity. The tyrosinase inhibitory activity was measured and the results are shown in Table 1. Arbutin was used as a positive control.

Sample Concentration (㎍/㎖) 125 250 500 1000 2000 Comparative Example 1 29.7±2.2 38.7±1.3 42.7±1.5 47.3±0.6 57.5±2.5 Comparative Example 2 12.1±1.3 19.5±1.5 28.9±0.9 32.1±0.8 35.7±2.2 Comparative Example 3 13.5±1.5 20.6±0.9 29.9±1.1 33.6±0.7 38.1±2.1 Example 1 59.7±0.8 64.7±0.9 82.9±0.7 88.7±0.8 90.4±2.1 Example 2 61.2±2.5 66.9±0.8 84.3±1.2 90.4±1.7 92.4±2.3 Example 3 61.7±2.2 68.3±0.4 85.1±0.8 92.1±0.9 93.9±2.1 Example 4 62.2±2.3 69.4±1.8 87.2±2.2 92.9±1.2 95.1±2.5 Arbutin 54.1±1.0 61.2±1.5 78.9±0.6 85.7±1.2 89.7±2.5

Looking at the results in Table 1, as a result of measuring the tyrosinase inhibitory activity of Comparative Examples 1. 2 and 3, the tyrosinase inhibitory activity was found to be concentration dependent depending on the treatment concentration from 125㎍/㎖ to 2000㎍/㎖, but the positive control group It was confirmed to have a low level of tyrosinase inhibitory activity when compared to arbutin. However, Examples 1 to 4 showed higher tyrosinase inhibitory activity than Arbutin.

In addition, Examples 3 and 4 using the hot water extract than the ethanol extract of Kakadu plum fruit showed higher tyrosinase inhibitory activity, and Examples 2 and 4, in which the content of the Kakadu plum fruit extract was increased to 5 parts by weight, were compared to Example 1, respectively. and Example 3, but the amount of increase was small, so it was found to be sufficient to use only a trace amount of 1 part by weight of the Kakadu plum fruit extract.

2) Measurement of melanin biosynthesis inhibitory activity

● Experimental method

B16F10 cells, a melanoma cell line, were purchased from the Korean Cell Line Bank, and Dulbecco's modified Eagle's containing 1% antibiotic (Gibco, USA) and 10% fetal bovine serum (FBS; Gibco, Grand Island, USA). Cultured in a 5% CO ₂ incubator at 37°C using DMEM medium, and subcultured once every two days.

B16F10 cells cultured in ^DMEM medium were inoculated at 2.0 After culturing for 24 hours in a 37°C, 5% CO ₂ incubator, samples are prepared by concentration. To stimulate samples and cells at each concentration, α-MSH, a stimulant, is mixed and added at 200 nM. After culturing in a 37°C, 5% CO ₂ incubator for 48 hours, the culture medium is removed and each well is washed with phosphate buffer (pH 7.4). Add 100㎕ of 1N NaOH to remove the cells and transfer them to a 1.5㎖ tube. After reacting at 80°C for 1 hour, measure the absorbance at 405nm in a spectrophotometer. Inhibition of melanin biosynthesis is indicated by the rate of decrease in absorbance of the sample solution with and without addition.

● Experiment results

The results of measuring melanin biosynthesis inhibitory activity are shown in Table 2 below. Arbutin was used as a positive control.

sample Melanin content (%) α-MSH(-) - α-MSH (+, 200 nm) 100 α-MSH (+, 200nM) + Comparative Example 1 50㎍/㎖ 44.2 ± 1.4 α-MSH (+, 200nM) + Comparative Example 2 50㎍/㎖ 65.9 ± 1.9 α-MSH (+, 200nM) + Comparative Example 3 50㎍/㎖ 62.2 ± 1.9 α-MSH (+, 200nM) + Example 1 50㎍/㎖ 15.3 ± 1.8 α-MSH (+, 200nM) + Example 2 50㎍/㎖ 14.1 ± 1.6 α-MSH (+, 200nM) + Example 3 50㎍/㎖ 13.8 ± 1.2 α-MSH (+, 200nM) + Example 4 50㎍/㎖ 12.7 ± 1.2 α-MSH (+, 200nM) + Arbutin 50㎍/㎖ 24.1 ± 1.4

Looking at the results in Table 2 above, the melanin biosynthesis inhibitory activity of Comparative Example 1. 2 and 3 was measured and showed a certain level of activity, but it was confirmed to have a low level of melanin biosynthesis inhibitory activity when compared to Arbutin, a positive control. It has been done. However, Examples 1 to 4 showed that Arbutin had a higher melanin biosynthesis inhibitory activity.

In addition, Examples 3 and 4 using hot water extracts rather than Kakadu plum fruit ethanol extracts showed higher melanin biosynthesis inhibitory activity, and Examples 2 and 4, in which the content of Kakadu plum fruit extract was increased to 5 parts by weight, were respectively Examples. Although it increased compared to Example 1 and Example 3, the amount of increase was so small that it was found to be sufficient to use only a trace amount of 1 part by weight of the Kakadu plum fruit extract.

<Test Example 2: Skin Moisturizing>

1. Cytotoxicity experiment

The cytotoxicity test was MTT (3-4) using Moseman's method (Moseman T J, Immuno Methods, 65, 55 (1983)) and Skaper et al.'s method (Skaper SD, et al, Cell Culture, 2, 17 (1990)). ,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) measurement method.

Human keratinocytes HaCaT were purchased from the Korean Cell Line Bank (Seoul, Korea). HaCaT was cultured in the laboratory using DMEM medium containing antibiotics of 10% (V/V) FBS, 100 U/mL of penicillin, and 100 g/mL of streptomycin in an animal cell incubator with 5% CO2 supply at 37°C.

Examples and Comparative Examples The compositions were dissolved in DMSO and added to the culture medium at 100 g/mL, and the cells were cultured in the culture medium for 24 hours. After treatment with MTT solution (5 mg/mL) and culturing for an additional 4 hours, the amount of formazan, which is a blue crystal, was measured by measuring the absorbance at 595 nm using an ELISA reader. Toxicity to cells was expressed as a percentage of the average absorbance value of the respective control group.

To investigate cytotoxicity in HaCaT keratinocytes, when treated at a concentration of 100 ㎍/mL, the degree of change in cell proliferation was calculated and investigated compared to treatment with only DMSO, which was the control group.

All results showed that the cell viability was 95-98%, and it was confirmed that both the Example and Comparative Example compositions had no cytotoxicity.

2. Evaluation of water retention capacity when applied to the human body

In order to evaluate the skin moisturizing effect of the compositions of Examples and Comparative Examples, the moisture retention capacity was measured on the skin to which each test substance of Examples and Comparative Examples was applied as follows. The test substance was used dissolved in purified water at 15% by weight.

To measure the moisture retention capacity, a certain amount (0.1g/4㎠) was applied to the inside of the forearm of 20 test subjects in a constant temperature and humidity room with an room temperature of 22℃ and a relative humidity of 45%, and the skin was measured before application, 1 hour after application, and 2 hours after application. The moisture content was measured. The measuring device used was a moisture content meter (corneometer CM820, Courage + Khazaka, Cologne, Germany), which measures the moisturizing power by measuring the skin's electrical capacity according to the skin's water content. The results are shown in Table 3 below.

electrical conductivity Before application 1 hour after application 2 hours after application Example 1 47 88 83 Example 2 47 90 85 Example 3 47 91 85 Example 4 47 93 88 Comparative Example 1 47 62 55 Comparative Example 2 47 54 51 Comparative Example 3 47 55 52

From Table 3 above, it was confirmed that the Examples according to the present invention had a significantly better moisture retention ability than the Comparative Examples, which proved that it was a material with an excellent moisturizing effect. In particular, the moisture retention effect was excellent in Examples 3 and 4 using the hot water extract of Kakadu plum.

3. Experiment on the effect of aquaporin-3 and involucrin synthesis promotion

In order to confirm the effect of increasing the synthesis of involucrin, which plays an important role in the barrier and moisturizing function of skin cells, and aquaporin-3, which plays an important role in water movement, the Example and Comparative Example compositions were applied to cells as follows, respectively. After processing, it was confirmed through Western blotting.

First, human keratinocytes HaCaT were inoculated, and 24 hours later, the example and comparative example compositions were added, respectively, and cultured in DMEM medium, 37°C, 5% CO2. After culturing for 24 hours, the cells were recovered, disrupted, and only the protein was extracted and stored at -40°C. The extracted protein was quantified using BCA saasy. The protein amount of each sample was diluted to 30 g, electrophoresed on a 12% SDS-gel, and then transferred to a PVDF membrane. After reacting with the primary antibody (rabbit anti-aquaporins-3 antibody), the remaining antibody was removed by washing and reacted with the secondary antibody (rabbit HRP-coupled anti-IgG antibody).

After removing the remaining antibody, a reagent (peroxidase substrate and chromogen solution) that reacts with the antibody to cause a color reaction was added. The developed band was measured and quantified through image analysis (BioRad, Universal Hood, USA), and the results are shown in Tables 4 and 5 below.

division Expression level of aquaporin-3 (%) control group 100 Example 1 158 Example 2 163 Example 3 165 Example 4 177 Comparative Example 1 121 Comparative Example 2 107 Comparative Example 3 110

As shown in Table 4, Comparative Examples 1, 2, and 3 had insignificant effects when compared to the control group, but Examples according to the present invention showed a synergistic effect of more than 50%, especially Example 3 and Example 3 using hot water extract. In Example 4, a remarkable synergistic effect was confirmed.

division Expression level of involucrin (%) control group 100 Example 1 158 Example 2 164 Example 3 164 Example 4 171 Comparative Example 1 124 Comparative Example 2 109 Comparative Example 3 111

When treated with HaCaT keratinocytes, the expression of involucrin, which plays an important role in skin barrier function and moisturization, was ineffective in Comparative Examples 1, 2, and 3 compared to the control group, as shown in Table 5 above, but the present invention The examples according to showed a synergistic effect of more than 50%, and in particular, a remarkable synergistic effect was confirmed in Examples 3 and 4 using hot water extract.

Through the above experimental results, it was confirmed that the composition of the present invention has excellent effects on skin barrier function and moisturizing.

<Test Example 3: Preventing skin wrinkles and maintaining elasticity>

1. Elastase inhibition rate measurement

The effect of inhibiting the activity of Elastase, an enzyme that decomposes elastin, was confirmed as follows.

Elastase derived from human white blood cells was used, and the synthetic substrate MeOSuc-Ala-Ala-Pro-Val-pNA was used as the elastase substrate. A 100mM Tris (pH 7.5) solution was used as the buffer solution. Elastase was used in a final amount of 0.2 mu using a buffer solution. In addition, the synthetic substrate of elastase was made into a 100mM solution using DMSO and then diluted using a buffer solution to reach a final concentration of 0.5mM.

At this time, the positive control was set to contain quercetin, known as an elastase inhibitor, at a concentration of 100 ppm. The samples of Examples and Comparative Examples were also added to 100ppm. The reaction was carried out in a 96-well plate and reacted at room temperature for 20 minutes. Absorbance was measured at 405 nm at 1-minute intervals using a spectrophotometer, and the slope of absorbance versus time was determined to be the enzyme activity. The elastase inhibition rate was calculated as follows and the results are shown in Table 6.

sample Elastase inhibition rate (%) Positive control group 65.18% Example 1 77.29% Example 2 78.21% Example 3 79.25% Example 4 82.15% Comparative Example 1 53.18% Comparative Example 2 41.28% Comparative Example 3 43.17%

As can be seen from the results in Table 6, the examples according to the present invention were found to have a remarkable elastase inhibitory ability that was superior to the positive control group, and the comparative examples also showed a certain degree of effect, but it was less effective than the examples of the present invention. In particular, Examples 3 and 4 using hot water extract showed even better effects.

2. Collagenase inhibition rate measurement

The effect of inhibiting the activity of collagenase, an enzyme that decomposes collagen, was confirmed as follows.

Collagenase derived from rat pancreas was used, and the synthetic substrate N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala was used as the collagenase substrate. The buffer solution was a solution containing 50mM Tricine, 10mM CaCl ₂ ·H ₂ O, and 400mM NaCl (pH 7.5). The synthetic substrate was dissolved to a concentration of 10mM using a buffer solution and then diluted to a final concentration of 75μM. Collagenase was dissolved at a concentration of 1 unit/ml using cold tertiary distilled water at 4°C and then diluted with a buffer solution to a final concentration of 0.05 unit/ml.

Samples of examples and comparative examples were tested at a final concentration of 100 ppm. At this time, the positive control group was epigallocatechin gallate (EGCG), which is known to have a collagenase inhibitory effect, and was set to a final concentration of 100 ppm. The reaction was carried out in a 96-well plate and reacted at room temperature for 10 minutes. Absorbance was measured at 345 nm at 1-minute intervals using a spectrophotometer, and the maximum slope of absorbance versus time was determined to be the enzyme activity. The collagenase inhibition rate was calculated as follows and the results are shown in Table 7.

sample Collagenase inhibition rate (%) Positive control group 57.12% Example 1 72.18% Example 2 75.12% 3 in action 74.42% Example 4 77.22% Comparative Example 1 49.15% Comparative Example 2 34.28% Comparative Example 3 35.71%

As can be seen from the results in Table 7 above, it was found that the examples according to the present invention had a remarkable collagenase inhibition ability that was superior to the positive control group, and the comparative examples also showed a certain degree of effect, but it was stronger than the examples according to the present invention. It was minimal, and in particular, Examples 3 and 4 using hot water extract showed a more excellent effect.

3. Measurement of cell proliferation effect

To confirm the activation of skin cell proliferation, the cell proliferation activity was measured using human fibroblasts.

After adding 10 μg/ml of the composition of Examples and Comparative Examples to the culture medium, human fibroblasts were cultured, MTT reduction was performed, formazan product was extracted with isopropyl ethanol, and absorbance was measured at 570 nm. By doing so, the cell proliferation activity was compared. At this time, culture medium without samples was used as a negative control. The measured results were calculated numerically using the formula below, and the results are shown in Table 8 below.

Cell proliferation ability (%) = absorbance in each sample group/absorbance in control group

sample Cell proliferation activity (%) Example 1 141.29% Example 2 144.14% Example 3 143.95% Example 4 146.24% Comparative Example 1 122.35% Comparative Example 2 113.12% Comparative Example 3 114.58%

As can be seen from the results in Table 8, the examples according to the present invention showed significantly superior cell proliferation activity compared to the cell proliferation activity (100%) of the untreated control group, and the comparative example also showed a certain degree of effect. However, it showed a significant difference from the example according to the present invention.

4. Measurement of collagen biosynthesis promotion effect

Fibroblasts were inoculated at a cell concentration of 5×10 ⁵ in IMDM medium supplemented with 10% FBS and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. After culturing for 24 hours, a diluted solution prepared by diluting the compositions of the examples and comparative examples in dimethyl sulfoxide to 500 ㎍/㎖ was added, cultured under the same conditions for 18 hours, and then UV irradiated for 1 hour to stress the cells. gave.

Afterwards, fibroblasts were recovered using Trizol reagent (invitrogen, USA), mRNA was extracted, and cDNA was synthesized through a series of processes. The PROCOLLAGEN TYPEⅠ gene region was amplified from the synthesized cDNA and the gene expression level was confirmed through electrophoresis. Gene amplification was performed using a thermal cycler (GenePro, Hangzhou bioer tech., CHINA) with 10x taq polymerase buffer, 10mM dNTP, 10 pool. Mix primer (F: AGC CAG CAG ATC GAG AAC AT, R: TCT TGT CCT TGG GGT TCT TG) and taq polymerase, add distilled water to adjust to 50㎕, 95℃ 5 minutes, 1 cycle/95℃ 1 minute/51℃ 2 Amplification was performed at 28 cycles per minute/72°C for 1 minute, and reaction was performed at 72°C for 5 minutes.

The expression rate of the generated procollagen was calculated according to the formula below. As a control group, normal fibroblasts that were not treated with samples or irradiated with UV were used. The results are shown in Table 9.

Procollagen expression rate (%) = B/A × 100 (%)

A: Procollagen expression level in the control group

B: Procollagen expression level in sample processed and UV-irradiated fibroblasts

sample Procollagen expression rate (%) Example 1 111.53% Example 2 113.65% Example 3 113.24% Example 4 115.68% Comparative Example 1 67.89% Comparative Example 2 41.25% Comparative Example 3 43.78%

As can be seen from the results in Table 9, the examples according to the present invention showed a significantly excellent effect of promoting collagen biosynthesis, and the comparative examples also showed a certain degree of effect, but showed a significant difference from the examples according to the present invention. .

As a result of the above review, it was found that the cosmetic composition according to the present invention is excellent in maintaining and improving skin elasticity and preventing skin wrinkles, and is ultimately the best cosmetic composition that can prevent skin aging.

Considering the above-mentioned test examples, the composition of the present invention is clinically effective for skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles by adding a small amount of Kakadu plum extract to multimolecular collagen with a low molecular weight and various molecular weight distribution. It has a very effective effect and is of great value as a cosmetic raw material.

Claims (5)

Multimolecular collagen with low molecular weight and diverse molecular weight distribution; and
Contains Kakadu plum fruit extract as an active ingredient, using water as the extraction solvent.
Skin moisturizing, skin whitening, maintaining and improving skin elasticity, and preventing skin wrinkles, characterized in that it consists of 95 to 99 parts by weight of the multimolecular collagen and 1 to 5 parts by weight of the Kakadu plum fruit extract, based on 100 parts by weight of the total cosmetic composition. A multifunctional cosmetic composition showing useful effects.
The multifunctional cosmetic composition according to claim 1, wherein the multimolecular collagen is low-molecular collagen with a molecular weight distribution of 200 to 5,000 Da.


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