KR101903750B1 - Method for Screening Cosmetics which Increase Moisturizing Ability of Skin - Google Patents

Abstract

The present invention relates to an evaluation method for evaluating the main ceramide components in the skin moisturizing ability and evaluating the skin moisturizing ability by comparing the composition ratio of CER [NS] with the skin moisturizing ability. Specifically, UPLC-MS / MS-ESI (CER [NS]) in human skin by quantitative determination of the ratio of CER [NS] in human keratin and the relationship with skin moisturizing ability (TEWL) And a method for screening a product that increases the moisturizing power by using the above-described moisture-imparting strength evaluation method.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for screening cosmetics,

The present invention relates to an evaluation method for evaluating the main ceramide components in the skin moisturizing ability and evaluating the skin moisturizing ability by comparing the composition ratio of CER [NS] with the skin moisturizing ability. Specifically, UPLC-MS / MS-ESI (CER [NS]) in human skin by quantitative determination of the ratio of CER [NS] in human keratin and the relationship with skin moisturizing ability (TEWL) And a method for screening a product that increases the moisturizing power by using the above-described moisture-imparting strength evaluation method.

The epidermis, which is the outermost layer of the skin, is an organ that is always in contact with the external environment and serves as a protective barrier. The skin barrier, which exists in the stratum corneum of the epidermis, suppresses the loss of water and electrolytes, protects the human body from external physical damage and chemicals, and prevents bacteria and viruses from penetrating into the skin. The stratum corneum consists of two components, lipid present between dead keratinocyte and keratinocyte, and lipid bilayer among these keratinocytes is known to play the most important role in maintaining skin barrier (Elias, PM, 1983 Epidermal lipids, barrier function, and desquamation J. Invest. Dermatol 80 : 44-49). The keratinocyte lipid is a lipid mixture of ceramides, cholesterol and free fatty acids, forming an intercellular lamellae.

Ceramide is a major lipid component of the keratin and plays an important role in the formation of the skin's moisture-retaining ability and skin barrier disease. Ceramide is a type of sphingoid lipid that has a structure in which fatty acids are linked to sphingosine or phytosphingosine. It accounts for about 40 ~ 50% of the intercellular lipid constituting the skin stratum corneum and is essential for showing the structure and function of the stratum corneum It acts as a lipid barrier to inhibit water evaporation in the stratum corneum and maintains a square structure of the stratum corneum.

Dryness of the skin is common in winter, but its etiology has not yet been fully described. Dry skin in the dictionary meaning refers only to lack or lack of moisture in the skin (less than 10%), clinically refers to rough skin with a slight erythema and cracks in the definition of dry skin and a scaly surface .

Dry skin is known to occur mainly in the upper layer of the epidermis, rather than the changes that occur throughout the skin, including the epidermis, dermis, and subcutaneous fat layer. Among the upper epidermis, changes occurring in the stratum corneum, It is thought to be the cause. Many studies have been carried out in terms of biochemical aspects of dry skin development. In particular, there are three factors, namely sebum, keratocyte lipolysis, natural moisturizing factor (NMF) in keratinocytes and degradation of desmosome The removal of keratinocytes is important, and skin becomes dry when such a normal biochemical process is interrupted.

The ceramide may be selected from the group consisting of sphingosine bases such as dihydrophosphingosine, sphingosine, phytosphingosine, 6-hydroxysingin and the like, and nonhydroxy fatty acids,? -Hydroxy fatty acids and ester-linked? -Hydroxy fatty acids Fatty acids. Since various sphingosine bases and fatty acids are combined to form ceramides, various types of ceramides may exist in the stratum corneum. However, quantitative and detailed studies on ceramides have not been conducted because of their analytical difficulties.

A variety of analytical methods have been used for identification and quantification of ceramides reported previously. (GC or GC-MS, Gaver RC, Sweeley, CC 1965. Methods for methanolysis of sphingolipids and direct determination of long-chain bases by Gas Chromatography, J. Am. Oil Chem . Soc. 42 : 294-8) , Thin layer chromatography (TLC, Weerheim, A., Pone, M., 2001. Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography. Arch. Dermatol . Res . 293: 191-9), high performance liquid chromatography (HPLC, Gaudin, K., Chaminade, P., Ferrier, D., Baillet, A., Tchapla, A., 1999. Analysis of commercial ceramides by non-aqueous reversed-phase liquid chromatography with evaporative light -scattering detection Chromatogr 49:... 241-8) , and enzyme or antibody-based assays (Edsall, LC, Spiegel, S. , 1999. enzymatic Measurement of Sphingosine 1-Phosphate Anal. Biochem . 272: 80-86) have been developed, but these methods have been disadvantageous in that they are non-specific, difficult to analyze in trace amounts, and consumed a lot of time. .... In recent years, mass analysis method in particular LC / MS (Lee, MH, GH Lee, JS Yoo 2003. Analysis of ceramides in cosmetics by reversed-phase liquid chromatography / electrospray ionization mass spectrometry with collisioninduced dissociation Rapid Commun Mass Spectrom 17: 64-75) or LC / MS / MS (Mano, N., Oda, Y., Yamada, K., Asakawa, N., Katayama, K., 1997. Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography / In this study, we have investigated the effects of ionic liquids on the physical properties of NPLC-ESI-MS (spray-ionization tandem mass spectrometry, Anal. Biochem. 244: 291-300) and Masukawa et al. The ceramides were analyzed by the spectrometry method. However, NPLC conditions are difficult to apply to general LC-MS (liquid chromatography-mass spectrometry) conditions, and RPLC-MS (reverse phase liquid chromatography-mass spectrometry) methods using mostly reversed phase columns are dominant. In recent years, there have been developed columns that can be used in reversed phase conditions with the characteristics of normal phase columns. Other conventional methods have limitations in providing quantitative reproducibility and precision in a complex sample matrix, and it has been difficult to simultaneously and rapidly analyze ceramides of various fatty acid chains.

CER [NS] (ceramide [nonhydroxy fatty acid sphingosine]), the binding chain between nonhydroxy fatty acids and sphingosine in various ceramides, (Novotny, J, B Janusova, M Novotny, A Hrabalek, K Vavrova, 2009. Short-chain ceramides decrease skin barrier properties. Skin Pharmacol . Physiol . 22: 22-30). In addition, CER [NS] accounts for 25% of the total ceramides present in the stratum corneum and can represent the constituent pattern of stratum ceramides (Ponec et al 2003).

Ultra Performance Liquid Chromatography (UPLC) has the advantage of separating multiple components effectively in a short period of time by using a fixed phase with a small particle size of 2μm or less. It has high resolution, high sensitivity and high peak capacity in a complex biological sample Can be quickly and accurately analyzed.

The present inventors have developed a quantitative method capable of quickly and accurately and selectively distinguishing the CER [NS] in the exudate by the fatty acid chain using the UPLC-MS / MS-ESI by the RPLC-MS method, The composition ratio was quantified. The results of the TEWL measurement and Principal Component Analysis were used to compare and analyze the results, and the present inventors completed the present invention by identifying major ceramide components in the skin moisturizing ability.

Accordingly, the present invention has developed a quantitative method capable of distinguishing the CER [NS] in the keratin by fast, accurate and selectively fatty acid chains, and quantitatively determining the composition ratio of CER [NS] in the human keratin to measure the TEWL And to provide a skin moisturizing evaluation method.

It is another object of the present invention to provide a method for screening a product that raises the moisturizing power by using the skin moisturizing evaluation method.

In order to achieve the above-mentioned object, the present invention provides a method for preparing a skin horny sample, comprising the steps of: a) obtaining a skin horny sample of a subject; b) measuring a transdermal moisture evaporation amount (TEWL) of the obtained keratinous sample; (C16Cer, C18Cer, C18: 1Cer, C20Cer, C24Cer, C24: 1Cer, C16SM and C18SM) contained in the keratin sample obtained in step a) using a chromatogram ; d) deriving a correlation function between the percutaneous moisture evaporation amount (TEWL) and each ceramide content through statistical analysis; And e) quantifying the skin moisturizing power of the subject based on the ceramide content in the skin keratin of the subject to be evaluated for skin moisturizing power, using the derived correlation function.

The present invention also provides a method for preparing a skin keratin sample, comprising the steps of: 1) obtaining a skin keratin sample, measuring the contents of short chain ceramides of C12 to C18 and long chain ceramides of C20 to C30 in the ceramide contained in the obtained keratin sample; 2) measuring the contents of short-chain ceramides of C12 to C18 and long-chain ceramides of C20 to C30 in the ceramides contained in the obtained keratinous sample, obtaining a skin keratin sample after applying the cosmetic composition to be tested to the skin; And 3) determining the moisturizing power of the applied cosmetic material from the change in the content of each of the short chain ceramides of C12 to C18 and the long chain ceramides of C20 to C30 measured in steps 1) and 2) And a method of screening a cosmetic material to be raised.

According to the method of the present invention, the composition ratio of CER [NS] among human keratin is determined by separating eight kinds of CER [NS] according to fatty acid length, and compared with the result of measurement of skin moisturizing ability (TEWL) The test method of the present invention can be utilized as a moisturizing evaluation method for evaluating skin moisturization and a method for screening a product that increases moisturizing power.

FIG. 1 is a schematic diagram showing the structure of eight ceramides C16Cer (CER [N (16) S (18)]), C18Cer (CER [N (18) S S (18)]), C20Cer (CER [N (20) S (18)]), C24Cer (18)), C16SM (([N (16) S (18)]) and C18SM (([N (18) S (18)].
Fig. 2 shows a calibration curve for the concentration of C16Cer (CER [N (16) S (18)]) and signal intensity (peak area ratio).
Figure 3 shows the amount of CER [NS] ceramides in human keratin.
Figure 4 shows the ratio of CER [NS] ceramides in human keratin.
Figure 5 shows the correlation analysis results using Pearson's correlation analysis between the C18Cer ratio and TEWL.
Figure 6 shows the correlation analysis results using Pearson correlation analysis between C20Cer ratio and TEWL.
FIG. 7 shows the principal component analysis results of the CER [NS] ratios of the volunteers obtained from the keratinous samples of healthy volunteers (N = 20).
FIG. 8 shows the relationship between the C16Cer and C24Cer ratios and the TEWL of the corneas of the volunteers.
FIG. 9 shows the relationship between the C20Cer ratio and the TEWL obtained from the upper part of the epiphysis of the additional volunteers (N = 14) and the statistical correlation analysis result using the Pearson correlation analysis.

The present invention relates to six CER [NS] ceramides (N-Palmitoyl-D- erythro- Sphingosine (C16 Ceramide, d18: 1/16 0), one to the N- stearoyl -D- erythro-sphingosine [N-stearoyl-D- erythro -Sphingosine (C18 Ceramide, d18: 1/18: 0)], N- oleoyl -D- erythro - sphingosine [N-Oleoyl-D- erythro -Sphingosine (C18: 1 Ceramide, d18: 1/18: 1 (9Z))], N- Araki diagram of one -D- erythro-sphingosine [N-Arachidoyl-D- erythro -Sphingosine (C20 Ceramide, d18: 1/20: 0)], N- one -D- erythro to league Nose-sphingosine [N-lignoceroyl-D- erythro -sphingosine (C24 Ceramide, d18: 1/24: 0)], N- four days Bono -D- erythro-sphingosine [N-nervonoyl-D- erythro -sphingosine (C24: 1 Ceramide, d18: 1/24: 1 (15Z))]) and include general formula (2) fatty acid chains specific ceramide that two kinds (N- palmitoyl -D- erythro-seuping tympanic phosphoryl choline [N-palmitoyl-D- erythro -sphingosylphosphorylcholine (C16SM, d18: 1/16: 0)], one by N- stearate -D-erit In-seuping tympanic phosphoryl choline [N-stearoyl-D- erythro -sphingosylphosphorylcholine (C18SM, d18: 1/18: 0)] of the total amount of ceramide from eight kinds of human keratinocytes and skin-friendly feature (TEWL) measurements, including The present invention provides a method for evaluating major ceramide components in skin moisturizing ability and evaluating skin moisturizing effect.

C24 ceramide (d18: 1/24: 0)

16: 0 SM (palmy tails sphingomyelin)

On the other hand, there are many ways to evaluate the moisturizing effect of the skin. The major categories are as follows: evaluation using skin cells, evaluation of moisturizing effect using animals, and evaluation of moisturizing effect in human body skin. Lipid analysis to quantify ceramide and the like The test is a general-purpose method that can be applied to both cell tests, animal tests, and human tests.

Types of evaluation method of moisturizing efficacy Kinds Method and content Cell test Horny type performance test
Skin cell differentiation factor identification test: FG, TGase,
Lipid biosynthetic enzyme activity and quantification test
Lipid analysis test: Ceramide quantitation Animal test Barrier resilience test: Reckless mice
Skin cell differentiation confirmation test: Immunohistochemical staining (H & E, FG, INV)
Lipid biosynthetic enzyme activity and quantification test
Lipid analysis test: Ceramide, cholesterol, etc. Clinical trial Measurement of moisture evaporation: TEWL
Moisture measurement: skin moisture meter (Corneometer)
Horny turn-over measurement test: DS
Keratinization test: D-squame®, Hi-scope
Stratum corneum lipid analysis test
Crustal layer sampling analysis (SSB): SEM, staining

In the present invention, in order to evaluate the skin moisturizing effect, a) a step of obtaining a skin horny sample of a subject; b) measuring a transdermal moisture evaporation amount (TEWL) of the obtained keratinous sample; (C16Cer, C18Cer, C18: 1Cer, C20Cer, C24Cer, C24: 1Cer, C16SM and C18SM) contained in the keratin sample obtained in step a) using a chromatogram ; d) deriving a correlation function between the percutaneous moisture evaporation amount (TEWL) and each ceramide content through statistical analysis; And e) quantifying the skin moisturizing power of the subject based on the content of ceramide in the keratin skin of the subject to be evaluated for skin moisturizing ability, using the derived correlation function.

In the present invention, a total of eight ceramides including CER [NS] ceramides (C16Cer, C18Cer, C18: 1Cer, C20Cer, C24Cer and C24: 1Cer) and two sphingomyelin ceramides (C16SM and C18SM) , And these ceramides are analyzed on a component-by-component basis using UPLC-MS / MS-ESI. Specific analytical conditions are described in Examples 1 and 2 below.

Ultra performance liquid chromatography (UPLC) enables the separation of multiple components in a short period of time by using a small particle size stationary phase of less than 2 μm. By using a mass spectrometer as a detector, selectivity and sensitivity can be increased.

Multiple reaction monitoring (MRM) mode, in which collision energies are applied to the parent ion to monitor the selected daughter ion at mass to charge ratio (m / z) after fragmentation, is selected Higher precision and lower detection limits are obtained because ions can be sampled more time for the mass to charge ratio (m / z) value of the ions to increase sensitivity and obtain signals that are not affected by interfering substances in the sample. do. Electrospray ionization (ESI) is the most commonly used ion source in LC-MS. Ceramide used in the present invention produced [M + H] ⁺ ion in positive ion mode and applied collision energy / MS induces fragmentation of CER [NS] ceramides with 264 masses of daughter ion and 184 mass of fragment ions with sphingomyelin ceramide.

Optimal separation conditions for each of the ceramides were determined using a C8 column, which is a reversed phase column, and a mobile phase of 5 mM ammonium formate / methanol (3/7, 0.1% formic acid) and 5 mM ammonium formate / isopropanol (7/3, 0.1% formic acid) The optimum shape and selectivity were shown when the composition was applied. Eight kinds of ceramides were effectively separated within 6 minutes.

In the present invention, the moisture content in the skin of a subject can be evaluated from the correlation function between the content of each ceramide and the transepidermal water loss (TEWL). Specifically, a ceramide having a short fatty acid has a negative effect on moisturizing due to a high polarity, and a ceramide having a long fatty acid can exhibit a positive effect due to a low polarity. In the ceramides analyzed in the present invention, C16cer and C18cer are short It belongs to the ceramide of the chain, and the other belongs to the long chain ceramide.

Therefore, when the content of C16cer and C18cer is high, the skin moisturizing ability can be evaluated to be low, and when the content of other ceramides is high, the skin moisturizing ability can be evaluated to be excellent.

The present invention also provides a method for preparing a skin keratin sample, comprising the steps of: 1) obtaining a skin keratin sample, measuring the contents of short chain ceramides of C12 to C18 and long chain ceramides of C20 to C30 in the ceramide contained in the obtained keratin sample; 2) measuring the contents of short-chain ceramides of C12 to C18 and long-chain ceramides of C20 to C30 in the ceramides contained in the obtained keratinous sample, obtaining a skin keratin sample after applying the cosmetic composition to be tested to the skin; And 3) determining the moisturizing power of the applied cosmetic material from the change in the content of each of the short chain ceramides of C12 to C18 and the long chain ceramides of C20 to C30 measured in steps 1) and 2) And a method of screening a cosmetic material to be raised.

The correlation between the content of each ceramide and the amount of percutaneous water evaporation was statistically analyzed. As a result, ceramide having less than C18 length had a positive correlation, ceramide having a C20 or longer length had a negative correlation with long chain ceramide and short chain ceramide The ratio plays a significant role in determining the skin moisturizing ability.

Therefore, it is possible to estimate the approximate skin moisturizing power from the ratio of the content of short chain ceramides of C18 or less, preferably of C12 to C18 and the content of C20 or more, preferably C20 to C30 long chain ceramide. In particular, if there is a decrease in short chain ceramides below C18 or an increase in long chain ceramides above C20 in the change of the contents of these ceramides after application of the cosmetic material, the cosmetic used may be evaluated as having increased skin moisturizing ability. It is possible to screen a product that raises the skin moisturizing power.

Hereinafter, the present invention will be described more specifically based on examples.

[Example 1] Concurrent analysis of eight ceramides

For analysis of CER (NS) (C16Cer, C18Cer, C18: 1Cer, C20Cer, C24Cer, C24: 1Cer) and Sphingomyelin (C16SM, C18SM), UPLC-MS is a Quattro Premier XE MS model connected to Waters AQUITY UPLC system Data were processed using Waters Masslynx 4.1 software.

(1) Analysis conditions

The respective analysis conditions are as follows.

- UPLC condition

Column: ACQUITY BEH C8 (2.1 mm x 50 mm, 1.7 m)

- Mobile:

A: 5 mM ammonium formate / methanol (3/7, 0.1% formic acid)

B: 5 mM ammonium formate in methanol / isopropanol (7/3, 0.1% formic acid)

Mobile phase conditions of UPLC-MS Time (minutes) Mobile (%) curve A B 0 20 80 6 0.5 20 80 6 2.5 0 100 6 4.5 0 100 6 6.0 20 80 One

Flow rate: 0.35 mL / min

Injection volume: 5 μL

Column temperature: 50 ° C

Automatic sample injector tray temperature: 4 ℃

Weak needle wash: 70% methanol

Strong needle wash: 100% methanol

MS analysis conditions Ionization mode ESI cation mode Source temperature (° C) 120 Desolvation temperature (캜) 350 Desolvated gas flow rate (L / h) 800 Cone gas flow rate (L / h) 100 Capillary voltage (kV) 3.2 Multiplier 700 Ion monitored MRM Collision gas (argon) Flow rate (mL / min) 0.25

MRM parameters Ceramides Selective ion (m / z) ^a Cone voltage (V) Collision energy C12Cer (IS) 482.3 > 264 20 18 C16Cer 538.4> 264 20 20 C18: 1Cer 564.6> 264 20 20 C18Cer 566.3> 264 20 22 C20Cer 594.5> 264 20 25 C24: 1Cer 648.6 > 264 20 22 C24Cer 650.5> 264 22 25 C16SM 703.6 > 184 20 24 C18SM 731.6> 184 20 24

^a selected ion for MRM, parent ion> fragment ion

(2) Preparation of internal standard solution and standard solution

Dilute the mixture with the internal standard solution was C12Cer the reference standard such that the methanol _{/ CHCl 3 (2:: 1} ) the concentration of 1000㎍ / mL dissolved in the mixed liquid following 20ng / mL to a concentration of methanol / CHCl ₃ (1 2) And used as an internal standard solution.

5 mg each of the standard products of CER [NS] (C12Cer, C16Cer, C18Cer, C18: 1Cer, C20Cer, C24Cer, C24Ser, C24SM, C18SM) and ceramide standard solution were precisely weighed and dissolved in methanol / CHCl ₃ 1) solution to give a concentration of 1000 μg / mL, and 100 μL of each standard solution was taken to a concentration of 100 μg / mL to prepare a standard mixed stock solution. The standard solution was diluted to the calibration curve (1, 5, 10, 20, 50, 100, 250 ng / mL)

[Example 2] Simultaneous analysis of 8 kinds of ceramide components

To verify the simultaneous analysis of ceramide six CER [NS] (C12Cer (internal standard substance), C16Cer, C18Cer, C18: 1Cer, C20Cer, C24Cer, C24: 1Cer) and sphingomyelin (C16SM, C18SM) Experiments on linearity, detection limit (LOD) and quantitative limit (LOQ) were carried out as follows.

1) Specificity (selectivity)

Chromatogram Whether the peak of the analyte is sufficiently separated from other interfering substances and can be accurately measured is confirmed from the chromatogram of the standard solution, the blank (blank D-squame tape extract) sample and the sample solution. It was confirmed that the peak of CER [NS] 7 ions and the ion peak of two kinds of sphingomyelin (C16SM, C18SM) were well detected without interfering with other components (FIG. 1).

2) Linearity

In order to examine the linearity of the quantitative concentration range at the time of the content test of the above-mentioned 8 kinds of ceramide components, a standard solution (using chloroform: methanol = 1: 2) was prepared by concentration, , And the results for C16cer are shown in FIG. The correlation coefficient of each component was more than 0.99 and linearity was confirmed. The correlation coefficient (R ² ) for each component is shown in Table 5.

3) Detection Limit (LOQ) and Quantification Limit (LOD)

The quantitative limit and the detection limit were calculated by comparing the S / N ratio of each peak. The detection limit was calculated based on S / N = 3 and the quantitative limit was calculated based on S / N = 10. (S / N ratio = 10) and the detection limit was 0.1 to 0.2 ng / mL (S / N ratio = 3) for each component, as shown in Table 5.

Ceramides Quantitation limits (ng / mL) Detection limits (ng / mL) Linearity (R ² ) C12 Cer (IS)
C16 Cer
C18: 1 Cer
C18 Cer
C20 Cer
C24: 1 Cer
C24 Cer
SM C16
SM C18 -
0.3
0.5
0.3
0.3
0.3
0.3
0.2
0.2 -
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1 -
0.9980
0.9956
0.9936
0.9936
0.9944
0.9952
0.9962
0.9944

[Example 3] Measurement of percutaneous moisture evaporation (TEWL) and extraction of keratin

Thirty-four healthy volunteers were divided into two groups of 20 and 14, respectively. The skin moisturizing capacity was measured by Trans-epithelial Water Loss (TEWL), and skin keratin was sampled at the same site. The test site was measured by placing a circle with a radius of 1.1 cm on the inside of the anterior chamber. As a measurement method, the inside of the anterior chamber was washed with soap, and it was adapted for 20 minutes in a constant temperature and humidity chamber (temperature 24 ± 2 ° C., relative humidity 40 ± 2%) and each measurement site was measured three times. Respectively. The measured value is expressed by the amount of moisture (g · m ² / h) evaporated per unit area. After the measurement of TEWL, D-squame standard tape was attached and 10 kernels were continuously taken from the same area using tape.

[Example 4] Ceramide analysis in keratin samples

(1) Analysis of protein content in keratinous samples

Each of the tapes was divided into two equal portions and used for protein quantification using 1/2 of keratinous sample tapes. Five 5-by-2 titer tapes were placed in 20 mL glass vials and 5 mL of 0.1% (w / v) sodium dodecyl cellite / 2% (w / v) propylene glycol PBS buffer solution was added and stirred for 60 minutes at room temperature. The supernatant was used as a sample solution by centrifugation (12,000 rpm, 4 ° C, 5 minutes). For quantification standard, a standard solution for calibration curve of 25 ~ 500 ㎍ / mL was prepared by using BSA of bicinchoninic acid (BCA) protein assay kit. 25 μL of each sample and standard solution was taken and analyzed for protein amount using BCA protein analysis method.

(2) Ceramide analysis in keratin samples

The other half of the keratinous sample tape was used for ceramide analysis in keratin samples. Put halve the tape 5 by one in 20mL glass vial methanol / CHCl ₃ (Final concentration: 12 ng / mL) was added to the mixture, and the mixture was centrifuged at room temperature for 30 minutes. Then, 3 mL of the supernatant was taken out and the mixture was centrifuged at 40 ° C and 30 ° C using a speed- Followed by drying under reduced pressure for several minutes. The dried samples were redissolved in methanol, shaken for 5 minutes, centrifuged (12,000 rpm, 4 ° C, 5 minutes) and the supernatant was analyzed with UPLC-MS / MS to obtain the result of ceramide content. Protein amount was corrected.

The content of each content is shown in Fig. In order to standardize the results of the ceramide content, the ceramide content was divided by the total CER [NS] content, and it was confirmed that each standard deviation was significantly reduced (FIG. 4). In the case of C18SM, the content was below or below the quantitative limit and was excluded from the content results.

[Example 5] Statistical analysis

(1) Pearson's correlation analysis

Correlation analysis identifies the degree and direction of the relationship between variables, and the degree of relationship between variables is the strength of the relationship. Correlation is expressed as a correlation coefficient (r), and a large correlation coefficient r means that there is a strong relationship between the two variables. The higher the absolute value of the correlation coefficient, the higher the relationship between the two variables, but the smaller the absolute value of the coefficient, the less the value is not significant. In the general statistical analysis, it can be said that the correlation is high when the correlation coefficient value is ± 0.7 to 0.9, and when there is a correlation value when the correlation coefficient value is ± 0.4 to 0.7, Can be interpreted as high.

The correlation between the ceramide-containing ratio and TEWL was evaluated by Pearson's method. It was confirmed that C18Cer had a positive correlation (Pearson's Coef = 0.414, p = 0.07) and C20Cer had a strong negative correlation (Pearson's Coef = -0.747, p = 0.00) (FIGS. The correlation results for each component are shown in Table 6.

Ceramides Pearson coefficient C16 Cer
C18: 1 Cer
C18 Cer
C20 Cer
C24: 1 Cer
C24 Cer
SM C16 0.262
0.02
0.414
-0.747
-0.346
-0.306
-0.117

(2) Principle Component Analysis (PCA)

Principal component analysis is the purpose of simplifying the dimension of reducing and summarizing multidimensional variables and analyzing the complex structure of variables that are generally correlated with each other. When there are p variables in the raw data, it is possible to explain a large part of the total variation with k main components smaller than p, and it is possible to interpret new data through the reduced main component, Statistical method. To do this, principal component analysis transforms variables and leads to new independent variables called main components. At this time, the order of importance can be considered on the basis of the size of the mutation possessed by each principal component. The major part of them is transformed so as to hold as much as possible of all the mutations inherent in the original data by several principal components. In other words, understanding the phenomenon by grasping the correlation structure, separating redundant information, reducing the dimension, and simplifying the variable structure (analytical structure) yields meaningful results in the population or system.

To investigate the relationship between each ceramide - containing ratio and TEWL, ceramide content ratio was analyzed by Principle Component Analysis (PCA) and statistical analysis was performed using the Minitab 14 program. Principal component 1 (PC1) obtained from the PCA analysis of each ceramide containing ratio accounted for 51% of the total variance of the ceramide content ratio and 16% of the main component 2 (PC2), indicating that PC1 and PC2 accounted for 68% of the total variation . When we plot the correlations between PC1 and PC2 for each applicant 's ceramide content ratio (Fig. 7), it was found that PC1 can roughly distinguish between applicants with low TEWL and high applicants. Note that the CER [NS] (C20Cer, C24Cer, and C24: 1Cer) of the long chain have a negative relationship with the major component 1 (PC1) ) (Fig. 8). C18: 1Cer was found to be in a very weak quantity relationship, and Sphingomyelin C16SM was found to be in a weak negative relationship, thus showing little influence on the correlation.

This is because ceramides having relatively short fatty acids have a negative effect on moisturizing due to high polarity, and ceramides having long fatty acids can exert a positive effect due to low polarity. This is consistent with previous studies that ceramides with long fatty acid chain structures can generate strong Van der Waals forces between molecules to form tighter molecular stacking (Munoz-Garcia et al 2008, Novotny et al.

Fourteen keratinous samples were analyzed to confirm the results obtained by using the coefficients of principal component 1 (PC1) obtained from the first 20 samples. The correlation between PC1 and TEWL obtained by calculating the ratio of each ceramide CER [NS] was evaluated, and a similar significant correlation was obtained as in the previous test. The long chain CER [NS] (C20Cer) (Pearson's coeff. = -0.598, p = 0.024, Fig. 10).

As a result, when the content of C16Cer and C18Cer, which are ceramides having a relatively short fatty acid among the ceramides present in the keratin of the skin, is high, the skin moisturizing ability is lowered, but the contents of ceramides C20Cer, C24Cer and C24: 1Cer having relatively long fatty acids Can be evaluated as having excellent skin moisturizing ability.

In addition, as described above, C16Cer and C18Cer in the ceramides contribute positively to skin moisture, C20Cer, C24Cer and C24: 1Cer contribute negatively, and C20Cer in particular has a strong negative correlation. In addition, since C18: 1Cer has a very weak amount and C16SM contributes to a weak negative relationship, the content ratio of ceramide present in the skin directly affects the moisture content rather than the total amount of the ceramide present in the skin.

Accordingly, the present invention can provide a method for measuring skin moisture resistance more accurately than by measuring the total amount of ceramide present in the skin by suggesting a correlation with the skin moisturizing power of the ceramide.

Claims (5)

(C24 Ceramide, d18: 1/20: 0), C24 Cer (C24 Ceramide, d18: 1/24: 0) and C24: 1 Measuring the respective contents of Cer (C24: 1 Ceramide, d18: 1/24: 1 (15Z));
(b) measuring the content of each of C20 Cer, C24 Cer and C24: 1 Cer among ceramides contained in the keratinocyte samples obtained by applying a cosmetic composition to be tested on the skin of the subject, ;
(c) The contents of C20 Cer, C24 Cer and C24: 1 Cer as measured in steps (a) and (b) were checked, and then the contents of C20 Cer, C24 Cer and C24: ; And
(d) evaluating the test cosmetic material reducing the content ratio of C20 Cer, C24 Cer and C24: 1 Cer as cosmetic materials for reducing the skin moisturizing ability of the subject based on the change in the content ratio determined in the step (c) And a step of evaluating the cosmetic material to be tested which increases the content ratio of each of C20 Cer, C24 Cer and C24: 1 Cer as a cosmetic material for increasing the skin moisturizing power of the subject. As a method,
The content ratios of the C20 Cer, C24 Cer and C24: 1 Cer are respectively the ratio of contents of C20 Cer, C24 Cer and C24: 1 Cer, based on the total content of CER [NS] ≪ / RTI >
delete [Claim 2] The cosmetic composition according to claim 1, wherein the cosmetic composition for increasing the content ratio of C20 Cer, C24 Cer and C24: 1 Cer has a ratio of each of C20 Cer, C24 Cer and C24: 1 Cer, And a negative correlation coefficient (r) value between the amount of percutaneous water evaporation of the horny sample. delete delete

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