KR102134930B1 - Method for estimating skin whitening effect of cosmetic composition - Google Patents

Method for estimating skin whitening effect of cosmetic composition Download PDF

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KR102134930B1
KR102134930B1 KR1020130159835A KR20130159835A KR102134930B1 KR 102134930 B1 KR102134930 B1 KR 102134930B1 KR 1020130159835 A KR1020130159835 A KR 1020130159835A KR 20130159835 A KR20130159835 A KR 20130159835A KR 102134930 B1 KR102134930 B1 KR 102134930B1
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melanin
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강혜리
박소현
김성우
조진훈
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Abstract

본 발명은 피부에 화장료 조성물을 도포한 후 각질층에 존재하는 멜라닌 양을 측정함으로써 화장료 조성물의 피부 미백 효과를 평가하는 방법이다.
본 발명에 따르면, 피험자에게 피부에 자극을 주는 과정과 긴 시험 기간의 부담을 덜 수 있을 뿐만 아니라 명확하고 객관적으로 화장료 조성물의 피부 미백 효과를 평가할 수 있다.
The present invention is a method of evaluating the skin whitening effect of a cosmetic composition by measuring the amount of melanin present in the stratum corneum after applying the cosmetic composition to the skin.
According to the present invention, it is possible to lessen the burden of the process of stimulating the skin and the long test period to the subject, and to clearly and objectively evaluate the skin whitening effect of the cosmetic composition.

Description

화장료 조성물의 피부 미백 효과 평가 방법{Method for estimating skin whitening effect of cosmetic composition}Method for estimating skin whitening effect of cosmetic composition

본 발명은 피부에 화장료 조성물을 도포한 후 각질층에 존재하는 멜라닌 양을 측정함으로써 화장료 조성물의 피부 미백 효과를 평가하는 방법이다. The present invention is a method of evaluating the skin whitening effect of a cosmetic composition by measuring the amount of melanin present in the stratum corneum after applying the cosmetic composition to the skin.

멜라닌은 피부, 털, 눈 등에 존재하는 흑갈색 알갱이 색소로서 멜라닌의 양에 따라 피부색이 결정된다. 또한 멜라닌은 일정량 이상의 자외선을 흡수하여 유해한 자외선이 인체 내로 침투하는 것을 차단하여 인체를 보호하는 역할을 한다. Melanin is a dark brown granule pigment present on the skin, hair, and eyes, and the skin color is determined by the amount of melanin. In addition, melanin absorbs more than a certain amount of ultraviolet rays and blocks harmful ultraviolet rays from entering the human body, thereby protecting the human body.

피부의 진피와 표피의 경계선 역할을 하는 기저막 위에 위치하는 멜라닌 형성 세포는 진피층에 위치한 섬유아세포의 영향을 받아 멜라닌을 생성한다. 멜라닌 형성 세포에서 생성된 멜라닌은 세포 돌기를 통해 주변의 각질 형성세포로 이동하여 피부에 넓게 분산됨으로써 자외선과 같은 유해 인자로부터 피부를 보호하고 피부색을 나타낸다. 정상 피부는 자외선과 같은 자극이 왔을 때 멜라닌 형성 세포가 평소보다 많은 멜라닌을 생성하고 분산하여 자외선이 피부 깊숙이 침투하는 것을 방지한다. 자극이 소멸하는 경우 멜라닌의 생성과 분산은 다시 정상 수준으로 돌아온다.
Melanin-forming cells located on the basement membrane, which act as a boundary between the dermis and epidermis of the skin, produce melanin under the influence of fibroblasts located in the dermal layer. Melanin produced by melanin-forming cells migrates to the surrounding keratinocytes through cell protrusions and is widely dispersed in the skin, thereby protecting the skin from harmful factors such as ultraviolet rays and displaying skin color. In normal skin, when stimulation such as ultraviolet rays comes, melanin-forming cells produce and disperse more melanin than usual, preventing ultraviolet rays from penetrating deep into the skin. When the irritation disappears, the production and dispersion of melanin return to normal levels.

한국인과 일본인과 같은 동양인들은 맑고 깨끗한 피부에 대한 관심이 높다. 이런 분위기를 반영하듯이 국내화장품업계뿐만 아니라 다국적 기업도 미백 원료 물질이나 효과에 연구가 증가하고 있다. Asians such as Koreans and Japanese are very interested in clear and clean skin. As reflected in this atmosphere, research on the whitening raw materials and effects is increasing not only in the domestic cosmetics industry but also in multinational companies.

미백 원료 물질로는 멜라닌 색소 생성에 중요한 작용을 하는 효소인 티로시나제를 억제하거나 항산화 작용을 가지는 원료들이 주로 사용되고 있다. As a whitening raw material, raw materials having an inhibitory effect or tyrosinase, an enzyme that plays an important role in the production of melanin pigment, are mainly used.

이에 화장료 조성물의 미백 효과를 평가하기 위해 티로시나제 활성억제를 평가하는 방법이 많이 사용되고 있다. 하지만 단지 시험관 상에서 티로시나제 활성 억제 능력을 가진 물질들이 멜라닌 세포를 대상으로 한 실험에서 더 나아가 임상에서 같은 정도의 효과를 나타낼지는 장담하기 어렵다. 또한 최근 티로시나제를 직접 억제하지 않더라도 우수한 미백효과를 나타내는 물질들이 많이 연구되고 있다.Therefore, in order to evaluate the whitening effect of the cosmetic composition, a method of evaluating tyrosinase activity inhibition has been widely used. However, it is difficult to guarantee whether substances with the ability to inhibit tyrosinase activity in vitro will have the same effect in clinical trials in melanocytes. In addition, many substances that exhibit excellent whitening effects have been studied even if tyrosinase is not directly inhibited.

따라서 티로시나제 억제효과만을 미백 기능성 화장품의 유효성 평가방법으로 사용하는 데에는 무리가 있으며, 새로운 평가기준의 개발이 시급한 실정이다. Therefore, it is difficult to use only the tyrosinase inhibitory effect as an evaluation method of whitening functional cosmetics, and the development of new evaluation standards is urgent.

미백 화장품의 미백 효능을 평가하는 임상 평가 방법으로 다음과 같은 방법이 알려져 있다. The following methods are known as clinical evaluation methods for evaluating the whitening efficacy of whitening cosmetics.

피험자의 피부에 인공적으로 자외선을 조사하여 색소 침착을 야기한 후에 시료를 도포하여 그 효능 여부를 결정하는 평가방법이 있다. 하지만, 자외선 조사에 의하여 피험자의 피부 표면에 수포가 발생할 정도의 과도한 홍반 반응이 발생할 수 있으며, 광알레르기 또는 광감작의 나타날 가능성도 배제할 수 없기 때문에, 시험에 참여하는 피험자에게는 부담이 되는 것이 사실이다. There is an evaluation method to determine whether or not the efficacy by applying a sample after causing the pigmentation by artificially irradiating ultraviolet rays on the subject's skin. However, due to ultraviolet irradiation, excessive erythema reaction to the extent that blisters may occur on the skin surface of the subject, and the possibility of photoallergy or photosensitization cannot be excluded, which is a burden for the subjects participating in the test. to be.

화장품의 미백 효능 평가는 기미, 점, 흑자, 검버섯과 같은 과색소침착증상을 가진 사람을 대상으로 이루어질 수도 있다. 하지만, 이러한 피부 과색소침착증은 그 발생 요인이나 기전이 자외선에 의한 멜라닌 생성 증가와 다르며, 비록 자외선에 의해 그 증상이 악화되는 경향은 있으나 자외선과 같은 자극이 없는 경우에도 멜라닌 생성과 분산이 계속적으로 증가한다. The evaluation of the whitening efficacy of cosmetics may be conducted for people with hyperpigmentation symptoms such as spots, dots, surpluses and blotch. However, such skin hyperpigmentation is different from the factor or mechanism of its increase in melanin production by ultraviolet rays, and although melanin production tends to worsen by ultraviolet rays, melanin production and dispersion continue even when there is no irritation such as ultraviolet rays. Increases.

또한 두 가지의 평가 방법은 피험자의 비교적 긴 기간 동안의 자율적인 화장품 도포를 전제로 하고 있으므로, 화장품의 도포가 평가자의 기준에 따라 적합하게 이루어지고 있는지 확인하기 힘들다. In addition, the two evaluation methods are based on the premise of autonomous application of cosmetics for a relatively long period of time, so it is difficult to confirm whether the application of cosmetics is properly performed according to the criteria of the evaluator.

이에, 시험의 부작용에 대한 피험자의 부담이 적으며, 화장품의 도포가 평가자에 의해 일관되게 이루어지고, 평가 기간이 짧으면서도 객관적인 결과를 도출해낼 수 있는 피부 미백 화장료 조성물에 대한 임상 평가 방법의 요구가 존재한다. Therefore, the burden of the test subject on the side effects of the test is small, the application of cosmetics is consistently made by the evaluator, and there is a need for a clinical evaluation method for a skin whitening cosmetic composition capable of eliciting an objective result with a short evaluation period. exist.

본 발명의 주된 과제는 피험자에게 피부에 자극을 주는 과정과 긴 시험 기간의 부담을 덜 수 있을 뿐만 아니라 명확하고 객관적으로 화장료 조성물의 피부 미백 효과를 평가할 수 있는 평가 방법을 제공하는 것이다. The main task of the present invention is to provide a method for evaluating the skin whitening effect of a cosmetic composition clearly and objectively, as well as to reduce the burden of a process and a long test period for irritating skin to a subject.

이를 위해 본 발명은To this end, the present invention

화장료 조성물의 피부 미백 효과를 평가하는 방법으로서,As a method for evaluating the skin whitening effect of the cosmetic composition,

화장료 조성물 도포 전과 도포 후 피부 각질층의 멜라닌 양을 측정함으로써 화장료 조성물의 피부 미백 효과를 평가하는 방법을 제공한다.It provides a method of evaluating the skin whitening effect of a cosmetic composition by measuring the amount of melanin in the stratum corneum layer before and after applying the cosmetic composition.

본 발명의 평가 방법에 의하면, 화장료 조성물이 누적 첩포 방법으로 도포되므로, 기존에 이루어지던 임상 평가에 비해 평가 기간이 단축되고, 평가자에 의한 일관된 도포가 이루어지기 때문에 피험자의 화장품 사용 습관이나 빈도 등에 따른 결과의 차이가 감소된다. 또한, 테이프 스트리핑 방법으로 평가 시료를 채취하기 때문에, 피험자의 부담이 적고, 누구든지 간단하게 평가하는 것이 가능해진다. 또한 각질층의 멜라닌 양을 측정하기 때문에 평가의 신뢰성을 증가시킬 수 있다. According to the evaluation method of the present invention, since the cosmetic composition is applied by the cumulative patch method, the evaluation period is shortened compared to the existing clinical evaluation, and the consistent application by the evaluator results in the cosmetic use habit or frequency of the subject. The difference in results is reduced. Moreover, since the evaluation sample is collected by the tape stripping method, the burden on the subject is small, and anyone can easily evaluate. In addition, since the amount of melanin in the stratum corneum is measured, reliability of evaluation can be increased.

도 1은 평가시험 전 비교제형 및 실험제형 도포 부위에서 채취한 각질층에 존재하는 멜라닌 양을 나타낸 도면이다.
도 2는 누적 첩포에 의한 비교제형 및 실험제형 도포 후 도포 부위에서 채취한 각질층에 존재하는 멜라닌 양을 나타낸 도면이다.
도 3은 비교제형 및 실험제형의 멜라닌 감소율을 비교하여 나타낸 도면이다.
1 is a view showing the amount of melanin present in the stratum corneum taken from the application site of the comparative formulation and the experimental formulation before the evaluation test.
2 is a view showing the amount of melanin present in the stratum corneum collected at the application site after application of the comparative formulation and the experimental formulation by cumulative patch.
3 is a view showing a comparison of the melanin reduction rate of the comparative formulation and the experimental formulation.

이하 본 발명을 더욱 상세히 설명한다. Hereinafter, the present invention will be described in more detail.

본 발명의 평가 방법은, 화장료 조성물의 피부 미백 효과를 평가하기 위해 피부 각질층의 멜라닌 양의 많고 적음을 지표로 하는 것을 특징으로 하고 있다. The evaluation method of the present invention is characterized in that the amount of melanin in the stratum corneum layer is large and low as an index to evaluate the skin whitening effect of the cosmetic composition.

멜라닌 생성이 억제된 피부는 그렇지 않은 피부에 비하여 멜라닌의 양이 유의하게 낮기 때문에, 각질층에 존재하는 멜라닌 양의 많고 적음을 지표로 하여, 간편하게 화장품의 미백 효능을 평가 할 수 있다.The skin in which melanin production is inhibited has a significantly lower amount of melanin than the skin in which it is not, so it is possible to easily evaluate the whitening efficacy of cosmetics by using a large and small amount of melanin present in the stratum corneum as an indicator.

이를 위해 화장료 조성물 도포 전과 도포 후 피부 각질층의 멜라닌 양을 측정한다. For this, the amount of melanin in the stratum corneum of the skin is measured before and after applying the cosmetic composition.

각질층은 피부의 가장 위에 있는 조직으로, 체외로부터의 이물질이나 자극으로부터 피부를 지키는 작용을 가지고 있다. The stratum corneum is the tissue on the top of the skin and has the function of protecting the skin from foreign substances and irritation from the outside of the body.

본 발명에 있어서의 평가 대상 부위는, 각질층을 입수할 수 있는 부분이라면 어떠한 부위도 포함될 수 있지만, 주된 부위로서는 전완 또는 등 부위를 들 수 있다.
The site to be evaluated in the present invention may include any site as long as it is a part where the stratum corneum can be obtained, but the main site may include a forearm or a back site.

본 발명에서 사용하는 화장료 조성물 도포 방법은, 누적 첩포 방법(Repeated Insult Patch Test)을 이용한다. 피험자의 전완 또는 등 부위에 48시간 첩포를 주 2회, 4주간 총 8회 적용하며, 화장료 도포 전과 도포 후 각질층을 채취한다. The method of applying the cosmetic composition used in the present invention uses a cumulative patch method (Repeated Insult Patch Test). A 48-hour patch is applied to the forearm or back of the subject twice a week for a total of 8 times for 4 weeks, and the stratum corneum is collected before and after applying the cosmetic.

각질층 채취는 테이프로 각질층의 표층 부분만을 채취하는 테이프 스트리핑 방법으로 간단하게 수행 할 수 있다. 이 때문에 사용자에게 부담을 주지 않고, 사용자의 피부에 대한 화장품의 미백 효능 여부를 평가할 수 있다. The stratum corneum collection can be performed simply by a tape stripping method that collects only the surface layer of the stratum corneum with tape. For this reason, it is possible to evaluate whether or not the whitening efficacy of cosmetics on the skin of the user is applied without burdening the user.

즉, 화장료 조성물이 도포된 부위에 스트리핑 테이프를 일정 시간, 즉 피부 각질층 채취에 충분한 시간 동안, 예를 들어 10초 내지 7분 동안 부착했다가 떼어냄으로써 피부 각질층을 채취할 수 있다. That is, the stratum corneum layer can be collected by attaching and peeling the stripping tape to the area where the cosmetic composition is applied for a predetermined time, that is, for a sufficient time for collecting the stratum corneum layer, for example, 10 seconds to 7 minutes.

이렇게 채취된 피부 각질층으로부터 멜라닌을 추출한다. 생화학적 방법, 예를 들면 동결 융해법, 초음파 파쇄법, 호모제네이트법(homogenate method)등을 매개로 하여 각 시료로부터 멜라닌을 추출한다. Melanin is extracted from the stratum corneum of the skin. Melanin is extracted from each sample through a biochemical method, such as a freeze-thawing method, an ultrasonic crushing method, and a homogenate method.

멜라닌의 양은, 종래부터 알려져 있는 방법으로 측정할 수 있다. 예를 들면, 멜라닌에 대한 항체와의 반응에 기초하는 효소 결합 면역 분석법(ELISA, Enzyme-linked immunosorbent assay), 방사면역분석법(radioimmunoassay), 웨스턴 블로팅(western blotting) 등의 방법을 이용 할 수 있다.
The amount of melanin can be measured by a conventionally known method. For example, methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and western blotting based on the reaction with an antibody to melanin can be used. .

본 발명의 평가 방법은 구체적으로,Specifically, the evaluation method of the present invention,

(S1) 피험자 각질층을 채취하여 각질층의 멜라닌 양을 측정하는 단계;(S1) collecting the subject's stratum corneum and measuring the amount of melanin in the stratum corneum;

(S2) 피부에 시험 대상 화장료 조성물을 도포한 후 피부 각질층을 채취하여 각질층의 멜라닌 양을 측정하는 단계; 및(S2) measuring the amount of melanin in the stratum corneum by applying the cosmetic composition to be tested on the skin and then collecting the stratum corneum of the skin; And

(S3) 상기 1) 단계 및 2) 단계에서 측정한 멜라닌양의 함량변화로부터 도포된 화장료 조성물의 피부 미백 효과를 평가하는 단계를 거친다.
(S3) A step of evaluating the skin whitening effect of the applied cosmetic composition is performed from the change in the amount of melanin measured in steps 1) and 2).

이때 시험 대상 화장료 조성물은 비교제형 화장료 조성물과 평가하고자 하는 실험제형 화장료 조성물을 사용할 수 있다. At this time, the cosmetic composition to be tested may be a comparative formulation cosmetic composition and an experimental formulation cosmetic composition to be evaluated.

각 화장료 조성물 도포 전후 측정된 멜라닌 감소율을 비교함으로써 실험제형 화장료 조성물의 피부 미백 효과를 객관적으로 평가할 수 있다.The skin whitening effect of the experimental formulation cosmetic composition can be objectively evaluated by comparing the melanin reduction rate measured before and after application of each cosmetic composition.

즉, 실험제형 화장료 조성물을 도포한 부위에서 측정된 멜라닌 감소율이 비교제형 화장료 조성물을 도포한 부위에서 측정된 멜라닌 감소율 보다 유의하게 높은 경우, 피부 미백 효능이 나타났다고 평가 할 수 있다.
That is, when the melanin reduction rate measured at the site where the experimental formulation cosmetic composition was applied is significantly higher than the melanin reduction rate measured at the site where the comparative formulation cosmetic composition is applied, it can be evaluated that skin whitening efficacy was exhibited.

본 발명의 평가 방법에 의하면, 화장료 조성물이 누적 첩포 방법으로 도포되므로, 기존에 이루어지던 임상 평가에 비해 평가 기간이 단축되고, 평가자에 의한 일관된 도포가 이루어지기 때문에 피험자의 화장품 사용 습관이나 빈도 등에 따른 결과의 차이가 감소된다. 또한, 테이프 스트리핑 방법으로 평가 시료를 채취하기 때문에, 피험자의 부담이 적고, 누구든지 간단하게 평가하는 것이 가능해진다. 또한 각질층의 멜라닌 양을 측정하기 때문에 평가의 신뢰성을 증가시킬 수 있다.
According to the evaluation method of the present invention, since the cosmetic composition is applied by the cumulative patch method, the evaluation period is shortened compared to the existing clinical evaluation, and the consistent application by the evaluator results in the cosmetic use habit or frequency of the subject. The difference in results is reduced. In addition, since the evaluation sample is collected by the tape stripping method, the burden on the subject is small, and anyone can easily evaluate. In addition, since the amount of melanin in the stratum corneum is measured, the reliability of evaluation can be increased.

바람직한 구현예로서, 본 발명에서 제시한 방법에 따라 화장품에 대한 피부 미백 효능을 분석하였다.
As a preferred embodiment, skin whitening efficacy for cosmetics was analyzed according to the method presented in the present invention.

[비교제형 및 실험제형 제조] [Production of comparative formulations and experimental formulations]

본 실험을 위하여 준비된 화장료 제형의 제법은 다음과 같다.The preparation method of the cosmetic formulation prepared for this experiment is as follows.

아래 표 1의 성분에 따라 수상, 유상을 75 ℃까지 가온하여 완전 용해한 후 아지 믹서(Agi Mixer)를 이용하여 수상을 스터링하면서 유상을 투입하였다. 유상 투입 후 5분간 3000rpm으로 아지 믹서로 혼합하였다. 혼합 후 중화 상을 투입하여 5분간 3000rpm으로 아지 믹서로 혼합하였다. 혼합 후 30 ℃까지 냉각하였다.According to the components in Table 1, the aqueous phase and the oil phase were heated to 75° C. to completely dissolve, and then the oil phase was added while stirring the water phase using an Agi Mixer. After the oil phase was added, the mixture was mixed with an azi mixer at 3000 rpm for 5 minutes. After mixing, the neutralization phase was added and mixed with an azi mixer at 3000 rpm for 5 minutes. After mixing, it was cooled to 30°C.

성분(중량%)Ingredient (% by weight) 비교제형Comparative formulation 실험제형Experimental Form 알부틴Arbutin -- 2.02.0 친유형 모노스테아린산글리세린Lipophilic glycerin monostearate 2.02.0 2.02.0 세테아릴알콜Cetearyl alcohol 2.02.0 2.02.0 스테아린산Stearic acid 1.51.5 1.51.5 폴리솔베이트 60Polysorbate 60 1.51.5 1.51.5 솔비탄스테아레이트Sorbitan stearate 0.60.6 0.60.6 하이드로제네이티드 폴리이소부텐Hydrogenated polyisobutene 1.01.0 1.01.0 스쿠알란Squalane 3.03.0 3.03.0 광물유Mineral oil 5.05.0 5.05.0 사이클로메치콘Cyclomethicone 5.05.0 5.05.0 디메치콘Dimethicone 1.01.0 1.01.0 초산토코페롤Tocopherol acetate 0.50.5 0.50.5 글리세린glycerin 5.05.0 5.05.0 베타인Betaine 3.03.0 3.03.0 트리에탄올아민Triethanolamine 1.01.0 1.01.0 산탄검Shotgun Sword 0.050.05 0.050.05 방부제antiseptic 적량Proper 적량Proper 정제수Purified water 잔량Balance 잔량Balance 합계Sum 100100 100100

[피험자 선정][Selected subjects]

18세~60세의 성인 남녀 중에서 평가 기관의 피험자 선정 기준에 만족하며, 선정 제외 기준에 해당되는 사항이 없는 사람을 피험자로 선정하였다. Persons between 18 and 60 years of age who were satisfied with the criteria for selecting subjects by the evaluation agency and who did not meet the criteria for selection exclusion were selected as subjects.

평가자는 ‘평가자가 피험자에게 알려주는 사항’을 피험자에게 설명하였고, 피험자는 자의에 따라 ‘임상시험 참가 동의서’를 작성하고 실험에 참여하였다. The evaluator explained'what the evaluator tells the subject' to the subject, and the subject prepared the'consent to participate in the clinical trial' at will and participated in the experiment.

[화장품 도포][Cosmetic application]

누적 첩포 방법을 이용하여 비교제형과 실험제형의 화장품이 피부에 도포되도록 하였다. 첩포 테이프는 8mm 직경의 Finn chamber를 사용하여 피험자의 전완 또는 등 부위에 48시간 부착하였다. 이러한 첩포 테이프를 4주간 주 2회, 총 8회 적용하도록 하였다.
By using the cumulative patch method, cosmetics of comparative and experimental formulations were applied to the skin. The patch tape was attached to the forearm or back of the subject for 48 hours using a Finn chamber of 8 mm diameter. This patch tape was applied twice a week for 4 weeks, 8 times in total.

[각질층 채취 방법][Keratin layer collection method]

피부로부터 첩포 테이프를 떼어낸 후, 각질층을 테이프 스트리핑 방법으로 박리 채취하였다. 채취 부위는 첩포 테이프에 의해 비교제형 및 실험제형이 도포된 부위로 하였다.
After removing the patch tape from the skin, the stratum corneum was peeled off by a tape stripping method. The collection site was defined as a site to which a comparative formulation and an experimental formulation were applied by a patch tape.

[피부 각질층 중 단백질 추출 방법] [Method of extracting protein from stratum corneum of skin]

각 각질층 테이프 1매에 대하여 500 ㎕의 T-PER Tissue Protein Extraction Reagent(Thermo Scientific 제조)를 넣고, 약 0.8 g의 유리 비드를 첨가하여 25분간 볼텍싱(vortexing)하여, 각질층을 파쇄하고, 각질층으로부터 단백질을 추출하였다.
500 μl of T-PER Tissue Protein Extraction Reagent (manufactured by Thermo Scientific) was added to each stratum corneum tape, and about 0.8 g of glass beads were added, followed by vortexing for 25 minutes to crush the stratum corneum and from the stratum corneum. Protein was extracted.

[피부 각질층 단백질의 정량][Quantity of skin stratum corneum protein]

회수한 단백질 용해액의 단백질량을 Pierce BCA Protein Assay Kit(Thermo Scientific 제조)를 사용하여 정량하였다.
The protein amount of the recovered protein lysate was quantified using the Pierce BCA Protein Assay Kit (manufactured by Thermo Scientific).

[멜라닌의 정량][Quantification of melanin]

상기 파쇄액으로부터, 멜라닌 양을 human melanin ELISA kit(Mybiosource 제조)를 사용하여 통상의 방법에 따라 측정하였다.
From the crushing solution, the amount of melanin was measured according to a conventional method using a human melanin ELISA kit (manufactured by Mybiosource).

[멜라닌 양 측정][Melanine amount measurement]

가. 비교제형 도포 부위의 멜라닌 양 측정end. Measurement of the amount of melanin at the application site of the comparative formulation

대조군 도포 부위에서 채취한 각질층에 존재하는 멜라닌 양을 도면 1에 나타내었다. The amount of melanin present in the stratum corneum collected at the control application site is shown in FIG. 1.

나. 실험제형 도포 부위의 멜라닌 양 측정I. Measurement of the amount of melanin at the site of application of the experimental formulation

미백 화장품 도포 부위에서 채취한 각질층에 존재하는 멜라닌 양을 도면 2에 나타내었다. Figure 2 shows the amount of melanin present in the stratum corneum collected at the whitening cosmetic application site.

다. 미백 화장품에 의한 피부 각질층의 멜라닌 억제 효능All. Melanin suppression effect of stratum corneum of skin by whitening cosmetics

비교제형 도포 부위에서 측정된 멜라닌 양과 실험제형 도포 부위에서 측정된 멜라닌 양을 비교하여 화장품으로 인한 멜라닌 생성 억제 정도를 도 3에 나타내었다.The amount of melanin measured at the application site of the comparative formulation and the amount of melanin measured at the application site of the experimental formulation are compared, and the degree of inhibition of melanin production due to cosmetics is shown in FIG. 3.

이상의 결과로부터 실험제형으로 인하여 피부 각질층의 멜라닌 양이 감소한 것을 알 수 있었다. From the above results, it was found that the amount of melanin in the stratum corneum layer was reduced due to the experimental formulation.

Claims (7)

(S1) 피험자 각질층을 테이프 스트리핑으로 채취하여 각질층의 멜라닌 양을 측정하는 단계;
(S2) 피험자 피부에 시험 대상 화장료 조성물을 누적 첩포 방법으로 도포한 후 피부 각질층을 채취하여 각질층의 멜라닌 양을 측정하는 단계; 및
(S3) 상기 (S1) 단계 및 (S2) 단계에서 측정한 멜라닌 양의 함량변화로부터 도포된 화장료 조성물의 피부 미백 효과를 평가하는 단계
를 포함하는 화장료 조성물의 피부 미백 효과 평가 방법.
(S1) measuring the amount of melanin in the stratum corneum by collecting the subject's stratum corneum by tape stripping;
(S2) measuring the amount of melanin in the stratum corneum by collecting the stratum corneum of the skin after applying the test subject cosmetic composition to the subject's skin by a cumulative patch method; And
(S3) evaluating the skin whitening effect of the applied cosmetic composition from the change in the amount of melanin measured in steps (S1) and (S2)
Method for evaluating skin whitening effect of a cosmetic composition comprising a.
삭제delete 제1항에 있어서, 상기 멜라닌 양은
효소 결합 면역 분석법, 방사면역분석법 또는 웨스턴 블로팅법을 통해 측정하는 것을 특징으로 하는 평가 방법.
The method of claim 1, wherein the amount of melanin
Evaluation method characterized by measuring by enzyme-linked immunoassay, radioimmunoassay, or Western blotting.
삭제delete 삭제delete 제1항에 있어서, 상기 시험 대상 화장료 조성물은
비교제형 화장료 조성물과 평가하고자 하는 실험제형 화장료 조성물을 사용하는 것을 특징으로 하는 평가방법.
According to claim 1, The cosmetic composition to be tested is
Evaluation method characterized by using a comparative formulation cosmetic composition and an experimental formulation cosmetic composition to be evaluated.
제1항에 있어서, 상기 피부 미백 효과를 평가하는 단계는
비교제형 화장료 조성물과 실험제형 화장료 조성물 도포 전후 멜라닌 감소율 비교를 통해 수행하는 것을 특징으로 하는 평가방법.
According to claim 1, Evaluating the skin whitening effect is
Evaluation method characterized in that it is performed through comparison of the melanin reduction rate before and after application of the comparative formulation cosmetic composition and the experimental formulation cosmetic composition.
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KR20090066824A (en) * 2007-12-20 2009-06-24 고려대학교 산학협력단 Compositions for skin whitening comprising puerarin or derivates thereof
KR101180258B1 (en) * 2010-06-09 2012-09-06 한불화장품주식회사 A skin-care agent containing Ophioglossum vulgatum extracts using microbial fermentation
KR101452061B1 (en) * 2012-01-10 2014-10-22 바이오스펙트럼 주식회사 Agents for skin whitening containing artemisinic acid, its derivatives and pharmaceutically acceptable salts thereof

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KR102544440B1 (en) 2022-06-09 2023-06-20 주식회사 제이투케이바이오 A novel anti-wrinkle evaluation method that treats Enterobacter aerogenes J2K-739 strain derived from skin flora and Enterobacter genus derived from skin flora as an irritant
KR102544441B1 (en) 2022-06-09 2023-06-20 주식회사 제이투케이바이오 A novel anti-inflammatory evaluation method that treats Enterobacter aerogenes J2K-739 strain derived from skin flora and Enterobacter genus derived from skin flora as an irritant

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