Cosmetic composition for improving skin condition containing peony extract as active ingredient for heatshock protein activation

Abstract

The present invention relates to a cosmetic composition which includes microcapsule particles (1 to 1,000 μm) stabilized by encapsulating a peony extract as an active ingredient, which is useful for skin, with a biocopolymer, and a fibrous protein surrounding the particles in a matrix form, a method of preparing the cosmetic composition, and the use of the cosmetic composition for improving a skin condition.

Claims

1. A cosmetic composition comprising microcapsules consisting of a biocopolymer encapsulating an active ingredient, a basic amino acid and a polyglycerly ester, and a fibrous protein surrounding each microcapsule in a matrix form to stablize the microcapsules, wherein the biocopolvmer and the basic amino acid are in a 1:1 weight ratio, and the biocopolymer and the fibrous protein are in a 1:1 weight ratio.

2. The cosmetic composition of claim 1, wherein the microcapsule has a size of 2 μm or less.

3. The cosmetic composition of claim 1. wherein the biocopolymer is one or more selected from the group consisting of an acrylate/acrylic acid copolymer; an acrylate/dimethicone copolymer; an acrylate/stearate-20 methacrylate copolymer; an aciylate/octylaciylamide copolymer; an acrylate/palmeth-25 acrylate copolymer; an acrylate copolymer; an acrylic acid/acrylonitrogens copolymer; an acrylamide/sodium acrylate copolymer; and an acrylamide/sodium acryloyldimethyltaurate copolymer.

4. The cosmetic composition of claim 1; wherein the basic amino acid is one or more selected from the group consisting of lysine; arginine; and histidine.

5. The cosmetic composition of claim 1, wherein the active ingredient comprises paeoniflorin or a cosmetologically acceptable salt thereof.

6. The cosmetic composition of claim 1; wherein the polyglyceryl ester is one or more selected from the group consisting of polyglyceryl stearate; polyglyceryl myristate; polyglyceryl laurate; polyglyceryl oleate; polyglyceryl isostearate; polyglyceryl distearate; and polyglyceryl tristearate.

7. The cosmetic composition of claim 1; wherein the fibrous protein is one or more selected from the group consisting of lecithin and gelatin.

8. The cosmetic composition of claim 1. wherein the composition comprises the biocopolymer and the polyglyceryl ester in a 1:5 weight ratio with respect to the total weight of the composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:

(2) FIG. 1 is a schematic diagram showing the flow chart and structure of a cosmetic composition according to one embodiment of the present invention;

(3) FIG. 2 is a set of microscopic images of cosmetic compositions having microcapsules and a matrix according to one example and comparative examples of the present invention;

(4) FIG. 3 is a graph showing the result of measuring expression levels of HSP-related signaling genes according to one embodiment of the present invention;

(5) FIG. 4 shows the result of measuring expression levels of HSPs according to a content according to one embodiment of the present invention;

(6) FIG. 5 shows the result of measuring expression levels of HSPs over time according to one embodiment of the present invention;

(7) FIG. 6 shows the result of an experiment of confirming the resistance of cells to oxidative stress due to HSP induction according to one embodiment of the present invention;

(8) FIG. 7 shows the result of an experiment of confirming the resistance of cells to oxidative stress due to HSP induction according to one embodiment of the present invention; and

(9) FIG. 8 is a schematic diagram showing the skin protective effect of the cosmetic composition prepared according to one embodiment of the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(10) Hereinafter, the present invention will be described in further detail with reference to the following examples. However, these examples are merely provided to exemplify the present invention, and the scope of the present invention is not limited by the following examples.

Preparation Example 1. Preparation of Peony Extract and Detection of Paeoniflorin

(11) 100 g of dried peony petals were cut into small pieces, added to and mixed with a 10-fold volume of water solvent (1 L), followed by soaking for 1 hour. While heating to 100° C., the mixture was extracted by being refluxed in a reflux condenser for 2 hours, and then subjected to second extraction for 1 hour. The extracted liquid was filtered with a gauze filter to remove a solid. After extraction, the resulting product was vacuum-dried. The final extracted solid was detected to be 0.02 g/L. The final extracted solid was dissolved at 1 mg/ml, and then analyzed by high performance liquid chromatography (HPLC), confirming that the concentration of the paeoniflorin contained in the solid was 0.93 mg/ml.

Preparation Example 2. Preparation of Microcapsule-Matrix Composition

(12) The schematic diagram of the prepared cosmetic composition is shown in FIG. 1. Specifically, 0.08 wt % each of an acrylate/acrylic acid copolymer and arginine were uniformly dispersed in distilled water and dissolved at 50° C. for 30 minutes. 0.08 wt % of the peony extract was added to the resulting solution, and then stirred at room temperature for 30 minutes. Afterward, 0.4 wt % of polyglyceryl oleate was added to the resulting product, and then stirred using a homogenizer at 2,000 to 8,000 RPM while cooling to 4° C., thereby forming microcapsules. For long-term stabilization and long-term prevention of aggregation, 0.09 wt % of hyaluronate was added to the formed microcapsules and slowly stirred at room temperature and a speed of 1,000 RPM or less, thereby forming a matrix. After the prepared cosmetic composition was observed using a microscope and compared with cosmetic composition of an example, the result is shown in FIG. 2. Comparative Examples 1 and 2 were prepared in the same manner as used in the example, except that components listed in Table 1 were used.

(13) As confirmed in FIGS. 1 and 2, it was seen that the cosmetic composition is a formulation containing microcapsules protecting the active ingredient and a matrix stabilizing the microcapsules.

(14) TABLE-US-00001 TABLE 1 (wt %) Comparative Comparative Comparative Component Use Example 1 Example 1 Example 2 Example 3 Distilled water Solvent 69.091 69.171 69.171 69.491 Paeoniflorin Active 0.0800 0.0800 0.0800 0.0800 ingredient Glycerin Thickener 14.714 14.714 14.714 14.714 Methyl Excipient 7.510 7.510 7.510 7.510 propanediol Butyl glycol Thickener 5.644 5.644 5.644 5.644 1,2-hexanediol Preservative 1.530 1.530 1.530 1.530 Allantoin Skin 0.100 0.100 0.100 0.100 conditioner Trehalose Skin 0.500 0.500 0.500 0.500 conditioner Glyceryl Biocopolymer 0.080 — 0.080 0.080 acrylate/acrylic acid copolymer Polyglycelyl- Polyglyceryl 0.400 0.400 0.400 — oleate ester Arginine Basic amino 0.080 0.080 — 0.080 acid Carbomer Thickener 0.181 0.181 0.181 0.181 Hyaluronate Fibrous 0.090 0.090 0.090 0.090 protein Total — 100 100 100 100

(15) Comparing Example 1, and Comparative Examples 1 and 2, in the formulation of Example 1, a clear microcapsule containing the active ingredient is shown under a microscope. However, in Comparative Examples 1 and 2, it was confirmed that the active ingredient is agglomerated with other components and then dispersed, without formation of microcapsules.

Experimental Example 1. Measurement of Stability of Formed Microcapsule-Matrix

(16) To measure the stability of the microcapsules, a composition was prepared in the same manner as in the examples, except that the hyaluronate of the hyaluronate-added composition (Example 1) was substituted with each of the same amounts of lecithin and gelatin, which are similar thickeners, and then the change in size of microcapsule particles was measured using a dynamic light scattering (DLS) device for 8 weeks. The result is shown in Table 2.

(17) TABLE-US-00002 TABLE 2 Experimental Change in particle size (average, μm) Change rate group 1 week 3 weeks 6 weeks 8 weeks (%) Control 1.38 Not Not Not Not measurable measurable measurable formed Hyaluronate 1.1  1.12 1.08 1.14 100.9 Lecithin 1.25 2.94 8.35 13.92  529.2 Gelatin 1.34 1.85 3.43 3.86 195.5

(18) As a control, Comparative Example 3 was used, and a composition of Comparative Example 3 was prepared in the same manner as in the examples, except that the components listed in Table 1 are used.

(19) One week after preparation, the size of the particles in Example 1 was 1.21 μm on average, and an error range was 0.34 μm.

(20) At one, three, six and eight weeks after formulation, the particle size was measured. When the measured particle is spherical, the size may be measured by calculating a flow rate (k) due to an electromagnetic field, but in the case of amorphous agglomeration, it was detected as unmeasurable because a measured value was not displayed. The rate of change is the change in expressed as a change rate of an average of values measured for 8 weeks with respect to the initial value in percentage (%).

(21) A control showed a completely unstable pattern since the spherical shape of a microcapsule was not observed after one week.

(22) When the microcapsules were stabilized with a hyaluronate, there was almost no change in size for 8 weeks, whereas when the microcapsules were stabilized with lecithin or gelatin, microcapsules were formed but considerably increased in size for 8 weeks so that they agglomerated, indicating instability.

Experimental Example 2. Expression Levels of HSP-Related Signaling Genes (Real-Time q-PCR)

(23) Human skin keratinocytes (BS cells) were obtained from the Korean Cell Line Bank (KCLB). For cell culture, cells were seeded at 10,000 cells/well in a 24-well plate as a cell culture dish containing a RPMI cell culture medium supplemented with 10% fetal bovine serum (FBS) and a 1% antibiotic, and then stabilized in an incubator for 24 hours at 37° C.

(24) The composition of Example 1 was inoculated in units of 100 μL on day 2, and after inoculation, the morphology of cells and related gene expression were examined. Specifically, the experiment was performed for controls (Comparative Examples 1 and 2) and the treatment group (Example). 100 μL of a sample per mL of the medium was treated for each group, and cultured for 24 hours at 37° C. and 5% CO.sub.2, followed by confirmation of whether a human HSP was expressed.

(25) As a stress condition, 100 μM H.sub.2O.sub.2 was additionally added to each 100 μL per mL of the medium, and then incubated for 24 hours.

(26) To check whether a human HSP was expressed or not, real-time gene amplification expression tests were conducted for HSP40, HSP90, HSP70 and HSP27 genes. Specifically, primers for each gene were constructed, the genes were amplified up to 49 cycles based on the primers using Taq polymerase activity, and the gene expression rates were detected in real time. The result is shown in FIG. 3.

(27) TABLE-US-00003 TABLE 3 SEQ ID NO: Name Sequence list Remark 1 HSP40 ggaggagctgttccatg 2 HSP90 ggagacctcgctat 3 HSP70 caccaagaagatgaaaatc 4 HSP27 acaagctctgcttatc

(28) TABLE-US-00004 TABLE 4 Control Treatment group Control Stress− Stress+ (Stress−) Stress+ Hsp40 Hsp90 Hsp70 Hsp27 Hsp40 Hsp90 Hsp70 Hsp27 100 283.45 174.35 212.4 244.75 174.56 256.87 331.46 441.54 384.54

(29) It was confirmed that each gene was expressed in the experimental environment without a problem, and first confirmed that there was no problem in terms of amplification efficiency over time. In addition, as confirmed in FIG. 3 and Table 4, it was seen that, under a stress condition (Oxidative stress: 100 μM H.sub.2O.sub.2-treated group), the total HSP expression increased approximately 2.8-fold, and according to the treatment with the example, a 1.5 to 2.4-fold higher gene expression rate than that under a stress condition, which is a stable expression rate, was shown. Such a gene expression rate was further amplified when stress was applied and the example was treated. The overall gene expression rate was amplified by 2.9 to 4.4-fold, confirming that the responsiveness to oxidative stress was increased approximately 2-fold, and particularly, it was confirmed that the expression rates of HSP70 and HSP27 genes, which are known to be important for the defense mechanism against oxidative stress, are greatly increased.

Experimental Example 3. HSP Expression Level (Western Blotting)

(30) 3-1. Expression Level According to Content

(31) Human skin keratinocytes (BS cells) were obtained from the Korean Cell Line Bank (KCLB). For cell culture, cells were seeded at 40,000 cells/well in a 6-well plate as a cell culture dish containing a RPMI cell culture medium supplemented with 10% fetal bovine serum (FBS) and a 1% antibiotic, and then stabilized in an incubator for 24 hours at 37° C. A sample was inoculated in units of 200 μl on day 2, and after inoculation, cell morphology and related gene expression were examined.

(32) An experiment was performed on controls (Comparative Examples 1 and 2) and the treatment group (Example 1). For each group, addition of 100 μL of a sample per mL of the medium was set as 10 wt %, and 10 to 50 wt % of the sample was added to the cells, and incubated for 24 hours at 37° C. in a 5% CO.sub.2 condition. One hour after incubation, the expression of a human HSP was detected. In this experiment, stress was not applied to the cells.

(33) Whether a change associated with human HSP expression affects a change in intracellular protein level was examined. Specifically, one hour after treatment of different contents (10 wt %, 20 wt %, 30 wt %, 40 wt % and 50 wt %) of a treated sample affecting HSP expression of the cosmetic composition of Example 1, 200 μL of cell lysis buffer was added to obtain cells, and then proteins were isolated at 100° C. for 10 minutes by adding 500 μL of mercaptoethanol. Proteins were separated according to a molecular weight by applying a charge to an SDS-PAGE environment for 3 hours, and detection was performed by observing the presence or absence of the protein by treating an HRP-tagged antibody. The result is shown in FIG. 4.

(34) As shown in FIG. 4, the change in extract content showed a slight increase in HSP27, but other HSPs did not show significant increases in expression level. These results confirm that the addition of DRBASE in a stress-free environment does not significantly affect cells.

(35) 3-2. Measurement of Change in Expression Level Over Time Depending on the Presence or Absence of Stress

(36) Human skin keratinocytes (BS cells) were obtained from the Korean Cell Line Bank (KCLB). For cell culture, cells were seeded at 40,000 cells/well in a 6-well plate as a cell culture dish containing a RPMI cell culture medium supplemented with 10% fetal bovine serum (FBS) and a 1% antibiotic, and then stabilized in an incubator for 24 hours at 37° C.

(37) A sample was inoculated in units of 200 μl on day 2, and after inoculation, cell morphology and related gene expression were examined. An experiment was performed on controls (Comparative Examples 1 and 2) and the treatment group (Example 1). For each experimental group, addition of 100 μL of a sample per mL of the medium was set as 10 wt %, and 10 to 50 wt % of the sample was added to the cells, and incubated for 24 hours at 37° C. in a 5% CO.sub.2 condition. One hour after incubation, the expression of human HSPs was detected. As a stress condition, 100 μM H.sub.2O.sub.2 was added to each 100 μL per mL of the medium and cultured for the same time. The protein change after treatment was detected in the same manner as the western blotting.

(38) To confirm whether there is a change in stability of a protein over the actual treatment time depending on the presence or absence of stress applied to cells, after oxidative stress (100 μM H.sub.2O.sub.2 treatment) was applied to cells under the same conditions, the intracellular concentration of HSPs was measured. The result is shown in FIG. 5.

(39) As seen in FIG. 5, HSPs drastically increasing after the application of oxidative stress showed a drastic decrease in the initial concentration after one and a half hours. The drastic decrease in HSPs was greatly increased when the cosmetic composition of an example was treated, and particularly, a significant effect of continuously maintaining a high HSP concentration at 30 minutes, which is the early state of the treatment, until about 3 hours was shown. As a result, in the case in which the cosmetic composition of the example was treated, but oxidative stress was not applied to cells, it was confirmed that it does not particularly affect HSP expression, whereas the treatment of the cosmetic composition of the example significantly maintains HSPs under an oxidative stress condition in which the HSP expression is rapidly reduced.

(40) Accordingly, it is considered that the example shows an insignificant expression-inducing effect on HSP under a stress-free condition, but shows a very potent HSP expression-inducing effect under an oxidative stress condition such as H.sub.2O.sub.2 treatment.

Experimental Example 4. Oxidative Stress-Induced Cell Aging

(41) It was confirmed whether the HSP induction increases resistance of cells against a high concentration of oxidative stress. Specifically, while increasing the concentration of oxidative stress, cells were cultured for 1 hour at 37° C. in 5% CO.sub.2 condition, and then cell viability was assessed by MTT assay. The result is shown in FIG. 6.

(42) As seen in FIG. 6, the cell viability that had no change at a low concentration of 50 μM showed significant toxicity from 200 mM, whereas in the group treated with the cosmetic composition of the example, it was confirmed that cells endured a high oxidative stress of 200 μM. It was confirmed that the treatment of the cosmetic composition of the example inhibits cell aging even when a low concentration of oxidative stress (100 mM) was treated for a long period of time.

(43) In addition, as confirmed in FIG. 7, after the application of oxidative stress, for 14 days, it was confirmed that the morphology of cells showing cell growth and senescence traits (white arrows) is significantly reduced in the group treated with the cosmetic composition of the example.

(44) The present invention relates to a cosmetic composition which includes microcapsule particles (1 to 1000 μm) which are stabilized by encapsulating a peony extract as an active ingredient, which is useful for skin, with a biocopolymer, and a fibrous protein surrounding the particles in a matrix form, a method of preparing the cosmetic composition and the use of the cosmetic composition for improving a skin condition.

(45) It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided they come within the scope of the appended claims and their equivalents