KR100566424B1 - The cosmetic compositions containing betulin for improving skin wrinkles - Google Patents

Abstract

The present invention relates to a skin wrinkle improvement cosmetic composition containing betulin, in detail, the cosmetic composition of the present invention is 0.0001 to 3% by weight, preferably 0.001 to 1% by weight of betulin to the total dry weight of the cosmetic composition It is characterized by containing in%.

Betulin according to the present invention exhibits an effect of inhibiting collagenase activity and an excellent effect of promoting cell regeneration and collagen production, and the wrinkle improving cosmetic composition containing the same also exhibits an excellent cell regeneration and collagen production promoting effect.

Betulin, wrinkle improvement cosmetic composition, collagen, collagenase, cell regeneration

Description

The cosmetic compositions containing betulin for improving skin wrinkles}

1 is a diagram showing thin layer chromatography of betulin and betulin standard (Betulin, SIGMA B-9757) of the present invention.

Figure 2 shows the IR spectrum of the betulin standard.

3 is a diagram showing the IR spectrum of the betulin of the present invention.

4 is a diagram showing the HPLC spectrum of the betulin standard (Betulin, SIGMA B-9757).

5 is a diagram showing the HPLC spectrum of the betulin of the present invention.

Figure 6 is a graph showing the comparison of the wrinkle improvement effect of the nutrition cream and the control of Formulation Example 3.

Figure 7a is a photograph of a replica (Replica) prepared for the test site of the subject before use of the control group.

Figure 7b is a photograph of a replica (Replica) prepared for the test site of the subject after use of the control group.

Figure 7c is a photograph of a replica (Replica) prepared for the test site of the subject before use of the nutrition cream of Formulation Example 3.

7D is a photograph of a replica prepared for a test site of a subject after using the nutrition cream of Formulation Example 3. FIG.

The present invention relates to a skin wrinkle improving cosmetic composition containing betulin of formula (1).

Cosmetics are deeply involved in our daily lives and used by many people. Before the development of human civilization, the purpose of cremation was to protect the body from nature.However, with the development of science and civilization, the purpose of cremation is to keep the body clean and beautiful, and to delay the aging to make a beautiful and pleasant life. Changed.

The function of cosmetics is not only clean and aesthetic functions, but also free radicals due to external factors such as moisture, pollutants and stress, ultraviolet rays, internal factors such as deterioration of immune cells and deterioration of cellular activity, It is to remove or suppress the cause of skin aging such as destruction of proteins, nucleic acids and cell membrane lipids by free radicals and peroxides. In addition, cosmetics having whitening, skin wrinkle improvement and suppression, and sunscreen functions to meet the consumer's desire for high-performance cosmetics continue to be developed. In particular, wrinkle improvement and suppression products are of great interest to consumers seeking to stay younger.

In general, as the skin ages, the skin loses its moisture, elasticity, color, and glossiness due to internal and external circumstances. This is called skin aging, and it is a process of increasing blemishes, wrinkles, and freckles. The skin of young people has good elasticity. In particular, children have a lot of collagen (Collagen) in the body is smooth. As you age, you may lose collagen or deteriorate, causing your skin to lose elasticity and wrinkles. Wrinkles of the skin are affected by various factors such as skin moisture content, collagen content, and immune function. Among them, collagen content is known to have the greatest effect.

Collagen is present in the dermal layer of the skin and accounts for about 70% of the total dry skin weight. The elasticity of the skin is known to be maintained by this collagen and elastin (elastic fiber). Collagen molecules are made in Fibroblasts and form triple helix structures after they are secreted out of the cell. The main functions of collagen are known to maintain the mechanical tightness of the skin, to strengthen the resistance of the connective tissue and the adhesion of the tissue, to sustain cell adhesion, induction of cell division and differentiation (Van der Rest et al., 1990). Such collagen is reduced due to the decrease of cellular activity due to natural aging and the increase of free radicals and free radicals caused by internal factors due to the activity of collagenase and external factors such as pollution, ultraviolet rays and stress (Ingrid Emerit et al., Free radical and aging, 1992).

In general, at 80 years of age, collagen is reduced by 65% compared to 20 years, resulting in thinner skin, which is known to be closely related to wrinkle formation of the skin (Arthur K. Balin et al., Aging and the skin, 1989). In recent years, extensive research on skin aging has emphasized the importance of collagen in the skin. When collagen metabolism is activated by the synthesis of collagen in the skin, the components of the dermal matrix are increased, and thus, the effects of wrinkle improvement, elasticity enhancement, skin strengthening and wound healing can be expected.

In order to obtain such an effect, conventionally, collagen was formulated into a cosmetic product. However, these cosmetics are to apply collagen to the surface of the skin, it is difficult to absorb the percutaneous absorption of collagen, a polymer, its function can not be expected. Recently, collagen is also injected directly into the dermis of the skin. However, this method is also difficult to expect correct skin wrinkles due to side effects.

In recent years, interest in substances that promote collagen synthesis has been increased to solve this problem, and collagen synthesis promoters include retinoic acid and proteins derived from animal placenta (Japanese Patent Laid-Open No. 8-231370). Became known. However, retinoic acid is unstable and complex formulation technology, and its use is limited due to safety problems such as irritation and redness of the skin, and protein from animal placenta is prohibited from using bovine extracts from mad cow disease. It is difficult to expect a skin wrinkle improvement effect by promoting collagen synthesis.

Therefore, there is an urgent need for the development of new collagen production promoters, which are safe for the living body, are easy to use, have high formulation stability, and have excellent collagen production promoting effects.

Therefore, the present inventors, while studying intensively to develop a new substance which is safe in vivo, has high formulation stability, and has excellent collagen production promoting effect, betulin, known as a substance having an anticancer and anti-inflammatory effect, inhibits collagenase activity. And it showed that the outstanding effect also in promoting collagen synthesis | combination, and also the stability at the time of compounding was discovered, and completed this invention.

An object of the present invention is to provide a wrinkle improvement cosmetic composition containing betulin.

The present invention relates to a skin wrinkle improving cosmetic composition comprising betulin.

The cosmetic composition of the present invention is characterized by including betulin at 0.0001 to 3% by weight, preferably 0.001 to 1% by weight based on the total dry weight of the cosmetic composition.

Betulin has been isolated from plants in Mexico and South America by Carl Djerassi (J.Am. Chem. Soc 78, 2312 ~ 2315 (1956)), and also contains data on Japanese organic compounds. SDBS (integrated spectral database system for organic compounds, http://www.aist.go.jp/RIODB/SDBS) is the base, and betulin is isolated from birch bark (Duke, JA, Writeups or information). summaries on approximately 2,000 economic plants, USDA, ARS, Beltsville, MD 20705) and anti-cancer and anti-inflammatory effects were also studied (Recio, M., et al. Investigation on the Steroidal anti-inflammatory activity of triterpenoids from Diospyros leucomelas, Planta) Medica, 61: 9 (1994). However, there was no study on the effect of betulin on promoting collagen production .

The skin wrinkle improvement cosmetic composition of the present invention is not particularly limited in the formulation, for example, it may have a formulation such as softening longevity, astringent longevity, nourishing longevity, nutrition cream, massage cream, essence, pack.

In addition, in the skin wrinkle improvement cosmetic composition of each formulation, other ingredients in addition to the above betulin can be appropriately selected and blended by those skilled in the art without difficulty according to the formulation or purpose of use of other cosmetics.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

Example 1 .

After extracting 1 kg of birch bark with 5 liters of ethanol, the extract was dried with a rotary vacuum condenser and dissolved by adding methanol. The solution was treated with activated charcoal, Whatman filter paper, GF / C filter paper and membrane filter paper (Millipore Co., Ltd.). Product), and then dried under a reduced pressure concentrator, and then suspended by adding purified water. This suspension was refrigerated to obtain a precipitate. This precipitate was dissolved in 90% methanol and separated by silica gel column chromatography to obtain betulin, a target compound.

Extracted and separated betulin was analyzed by comparison with betulin standard (Sigma Reagent B 9757).

(1) Thin layer chromatography:

1. Betulin standard (Betulin, SIGMA B-9757) 2. Extracted betulin sample

3. Betulin standard (Betulin, SIGMA B-9757) 4. Extracted betulin sample

5. Betulin Standard (Betulin, SIGMA B-9757) 6. Extracted Betulin Samples

Conditions: TLC, stationary phase: silica gel 60 F ₂₅₄ (MERCK 1.05554),

Mobile phase: N-nucleic acid (normal nucleic acid): ethyl acetate (2: 1),

Color development: heating after spraying 10% sulfuric acid

(2) FT-IR (JASCO FT-IR 410, KBr Disc)

(3) High Performance Liquid Chromatography (HPLC)

Condition: System: Waters 486 UV 205nm

Column: Capsule pak C ₁₈ UG120 4.6 × 250 mm (Shiseido),

Mobile phase: 90% methanol,

Temperature: 40 ℃,

Injection Vol.: 20μl

Flow rate: 1.0 ml / min

Run time: 10 minutes

(4) Melting Point Meter (Electrotherma 9300)

The results of the TLC, IR, HPLC, melting point of the target compound are as follows.

TLC (Rf value) FT-IR HPLC Melting point Standard material Extracted betulin Standard material Extracted betulin Standard material Extracted betulin Standard material Extracted betulin   0.32 0.32 3444.2 3075.9 2942.8 2870.5 1644.0 1483.9 1453.1 1389.4 1374.0 1336.4 1301.7 1245.7 1188.9 1136.8 1105.9 1081.8 1035.5 1009.5 983.5 971.9 879.3 545.7 3440.3 3073.0 2942.8 2870.5 1643.0 1483.9 1453.1 1387.5 1375.0 1343.1 1298.8 1246.7 1188.9 1132.9 1105.9 1080.9 1033.6 1009.5 983.5 971.9 879.3 545.7 Retention time: 8.08 minutes                                                  Peak area: 3203076.8 Retention time: 8.06 minutes                                                  Peak area: 3069795.9                                                  Purity: 94% 223 ~ 225 ℃ 223 ~ 225 ℃

1 shows thin layer chromatography of betulin and betulin standard of the present invention (Betulin, SIGMA B-9757), and FIGS. 2 and 3 show IR spectra of betulin standard and betulin of the present invention, respectively. . 4 and 5 show HPLC spectra of betulin standards and betulin of the invention, respectively.

As shown in Table 1 and FIG. 1, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and FT-IR results are the same as those of betulin standard (Sigma B-9757). It was found that the ingredient extracted from the shell was betulin.

Experimental Example 1.

The effect of collagenase activity inhibition on betulin was measured.

1) Experiment Method

Collagenase activity inhibitory effect experiment was carried out in the following manner.

0.1 mg of collagenase (SIGMA C5138) is dissolved in 1 ml of 0.1 M Tris Buffer (pH 7.1), and 0.5 mg of collagen (SIGMA C9879) is dissolved in 1 ml of 0.1 M Tris Buffer (pH 7.1). After the preparation, samples were prepared for each concentration. 0.05 ml of the prepared sample, 0.05 ml of collagenase and 0.4 ml of collagen were administered to the test tube and reacted in a 37 ° C. water bath for 30 minutes, and then 1 ml of 25 mM citric acid solution was added to terminate the reaction. After the reaction was completed, 5 ml of ethyl acetate was injected into the test tube, shaken, and centrifuged at 3000 rpm for 5 minutes, and the absorbance of the ethyl acetate layer was measured at 320 nm. The results of the six experiments were used as data.

2) Experiment result

Collagenase activity inhibitory effect was calculated by the following formula, the results are shown in Table 2 below.

Concentration (μg / ml) Collagenase activity inhibition rate (%) Collagenase Activity 50% Inhibitory Concentration (µg / mL) 5 18.3 12.19 15 62.8 20 89.5 25 95.6

As can be seen from the above experimental results, betulin concentration of 25 ㎍ / ㎖ was inhibited collagenase activity of more than 90%, the concentration that inhibits the collagenase activity 50% was 12.19 ㎍ / ㎖, even at very low concentration It was confirmed that the excellent collagenase activity inhibitory effect.

Experimental Example 2.

The effect of cell proliferation using cell culture on betulin was measured.

1) Experiment Method

Cytotoxicity and proliferation experiments were carried out in the following manner. Incubate the cells (fibroblast, 3T3) in 96-well microplates at 5,000 cells / well and incubate in a thermostat for 30 minutes and sample the concentration (0.5, 5, 50, 100, 200, 400 (µg / ml, respectively). )) And incubated for 72 hours. Thiazoline blue was administered and further cultured for 4 hours. The culture medium was discarded and the reaction stopper was added to each well of the microplate, and stirred for 5 minutes, and then absorbance was measured at 570 nm. As a control group, 10% Fetal Bovine Serum (FBS) medium was used as a sample injection amount to co-culture as an optimal condition for cell growth. The cell proliferation of the control group was 100% and the cell proliferation rate of the sample injection group was calculated. . The results of the six experiments were used as data. The results are shown in Table 3 below.

2) Experiment result

The cell proliferation effect was calculated by the following formula, and the results are shown in Table 3 below.

Betulin concentration (µg / mL) Cell proliferation rate (%) Control 100 0.5 107.52 5 116.36 50 117.20 100 132.03 200 154.30 400 193.01

As shown in Table 3, when the cell proliferation at the optimal cell growth condition was 100%, the cell proliferation effect was 193% at the betulin concentration of 400 µg / ml. In addition, having a cell proliferation effect means that betulin is not toxic to cells, and from the cell proliferation effect experiment, it was confirmed that the cell proliferation effect of the betulin of the present invention is not cytotoxic and safe.

Experimental Example 3.

The effect of promoting collagen production using cell culture was measured for betulin.

1) Experiment Method

Collagen production promoting effect was measured by the method of Crystal Violet assay. This method is to examine the effect on the cell proliferation. This experiment is to examine the cell proliferation and protein synthesis ability through this method. Crystal violet dye is a reagent that binds to proteins and uses these properties to measure cell protein synthesis and collagen synthesis. This experimental method was as follows.

Cells were tested using Swiss Albino mouse cells (3T6 Fibroblast). Dispense 5,000 / well 3T6 Fibroblast into a 96 well microplate and, after 24 hours, sample concentrations (0.5, 5, 50, 100, 200, 400 (each). Μg / ml)), and incubated in a 5% CO ₂ incubator for 72 hours, then carefully removing the culture medium and washing the 96 well microplates with saline-phosphate buffer (PBS solution), at 37 ° C. for 30 minutes. Dried. After adding the crystal violet reagent and staining at room temperature for 5 minutes and washing the dye solution well with distilled water, the combined dye was dissolved by adding 0.2M Phosphoric acid and ethanol, and the absorbance was measured at 590 nm after 5 minutes. It was. As a control group, 10% fetal bovine serum (FBS) medium was administered as the sample injection amount, and co-culture was performed under the optimal conditions for cell growth. The collagen synthesis of the control group was 100%, and the collagen synthesis of the sample injection experiment group was calculated. The results of the six experiments were used as data. The results are shown in Table 4 below.

2) Test result

Collagen production promoting effect was calculated by the following formula, the results are shown in Table 4 below.

Betulin concentration (µg / mL) Collagen production promotion rate (%) Control 100 0.5 115.58 5 121.02 50 121.93 100 139.20 200 182.85 400 226.47

As shown in Table 4, when collagen synthesis was 100% at the optimum cell growth conditions, the collagen synthesis effect was 226% at the betulin concentration of 400 µg / ml. Therefore, it can be seen from the above experimental results that the betulin of the present invention has excellent collagen synthesis promoting effect.

From the above experimental results, it was confirmed that betulin inhibits the activity of collagenase related to the skin wrinkles, excellent cell proliferation effect, and excellent collagen synthesis promoting effect. Therefore, it can be seen that betulin of the present invention has excellent collagenase activity inhibitory effect, cell regeneration and collagen production promoting effect.

Experimental Example 4.

Wrinkle improvement effect of nutrition cream containing the betulin of Formulation Example 3 was measured.

1) Experiment Method

10 females over 35 years of age were divided into left and right faces, divided into the nutrition cream (product containing betulin) and the control group (product containing no betulin) of prescription example 3, and 0.2g for 2 months for 2 months. It was applied to an area of 2 cm 2. Subjects were treated with silicone (Silflo; Flexico Ltd., England) in a constant temperature and humidity (22 ± 2 ° C, 50 ± 5% Humidity) chamber, respectively, prior to using the nutrition cream or control of Formulation Example 3 and two months after application. A replica of a test site of was prepared. Replica produced in this way was analyzed using Skin Visiometer (model name: SV600, manufacturer: Courage + Khazaka electronic GmbH, Germany), a computer imaging system that measures the depth of wrinkles, and the result was R1 ~ R5. It is expressed as a value of. Here, R1 to R3 represent the degree of deep wrinkles, and R4 to R5 represent the degree of skin roughness.

2) Experiment result

Wrinkle reduction rate was calculated by substituting the values of R1 to R5 measured in the following equation.

Wrinkle reduction rate (%) = [(measured value before use-measured value after 2 months application) / measured value before use] × 100

The reduction ratios of R1 to R5 for the subjects calculated by the above formulas are shown in Tables 5 and 6, and the significance for the control group in Table 6 was p <0.05 as Sigmastat version 2.03 (manufactured by SPSS Inc.). Assay at level (95% confidence interval). In addition, the average value of the reduction rate of R1 ~ R5 described in Table 5 and 6 in a graph showing the wrinkle improvement effect of the nutrition cream of Formulation Example 3 compared to the control (Fig. 6), the photo of the replica (Replca) control Or shown in Figures 7a, 7b, 7c and 7d divided into before and after the use of the nutrition cream of Formulation Example 3.

Wrinkle Reduction Rate by Control Decrease in R1 Decrease in R2 Decrease in R3 Decrease in R4 R5 reduction rate Subject 1 8.21 6.78 6.67 9.52 12.12 Subject 2 3.08 6.51 6.21 11.91 14.29 Subject 3 6.94 6.53 8.18 14.29 7.90 Subject 4 8.29 7.45 11.11 16.00 14.63 Subject 5 8.90 6.43 8.03 6.45 11.11 Subject 6 7.84 7.90 12.90 0.00 5.88 Subject 7 7.29 8.38 9.16 8.86 13.79 Subject 8 7.18 8.98 6.52 13.04 17.50 Subject 9 11.36 9.09 10.67 16.67 15.39 Subject 10 9.33 7.58 9.68 14.29 16.67 Average 7.84 7.56 8.91 11.10 12.93 S.Error 0.67 0.32 0.70 1.61 1.18

Wrinkle Reduction Rate by Nutritional Cream of Formulation Example 3 Decrease in R1 Decrease in R2 Decrease in R3 Decrease in R4 Decrease in R5 Subject 1 41.57 41.98 42.86 41.18 28.57 Subject 2 52.81 53.70 41.24 44.74 35.29 Subject 3 51.04 51.87 48.52 37.50 28.57 Subject 4 48.98 49.47 46.15 42.11 30.77 Subject 5 42.21 42.95 34.67 32.14 20.00 Subject 6 9.13 13.92 15.39 11.11 18.52 Subject 7 11.70 11.96 4.90 0.00 20.59 Subject 8 2.78 3.41 0.00 0.00 38.89 Subject 9 36.00 14.29 34.72 33.33 17.86 Subject 10 49.76 50.98 41.11 37.50 20.59 Average 34.60 * 33.45 * 30.96 * 27.96 * 25.96 * S.Error 6.09 6.32 5.58 5.51 2.37

*: Significance test with control group (p <0.05)

As shown in Tables 5 and 6 and FIGS. 6 and 7A, 7B, 7C, and 7D, the deep wrinkles (R1 to R3) are applied when the nutrition cream of Formulation Example 3 is applied to the skin, as compared to the case where the control is applied to the skin. ) And decrease in skin roughness (R4 ~ R5) can be seen clearly. From these results, it was confirmed that the wrinkle improvement effect of the cosmetic composition containing betulin was very excellent.

Experimental Example 5.

About the cosmetic composition containing betulin of the following prescription examples 1, 2, and 3, the stability was confirmed on the conditions of room temperature, refrigeration, 40 degreeC, and 60 degreeC for about 5 months. As a result, there was no change in cosmetic properties.

From this, it was confirmed that the cosmetic composition containing the birch extract of the present invention is stable.

Prescription Example 1.

The prescription example of the softening longevity (skin) in the cosmetic containing the betulin of Example 1 of this invention is as follows.

ingredient Content (% by weight) Betulin 0.5 Butylene glycol 4.0 glycerin 8.0 Carboxy Vinyl Polymer 0.06 Disodium ethylenediaminetetraacetic acid 0.01 ethanol 7.0 Triethanolamine 0.2 Sorbitan monostearate 0.8 Hydrogenated Castor Oil 0.4 Phenyltrimethicone 3.5 Paraoxybenzoic acid propyl 0.2 Methyl paraoxybenzoate 0.2 Purified water Remaining amount system 100.0

Prescription Example 2.

The prescription example of the nutritional cosmetics (milk lotion) in the cosmetics containing the betulin of Example 1 of this invention is as follows.

ingredient Content (% by weight) Betulin 0.5 Stearic acid 1.0 Cetyl alcohol 1.0 Glyceryl Monostearate 1.0 Polyoxyethylene sorbitan monostearate 1.0 Sorbitan sesquioleate 1.0 Liquid paraffin 4.0 Squalane 3.0 Carlylic / Capric Triglycerides 4.0 Carboxy Vinyl Polymer 0.1 Butylene glycol 5.0 Triethanolamine 0.2 Paraoxybenzoic acid propyl 0.2 Methyl paraoxybenzoate 0.2 Purified water Remaining amount system 100.0

Prescription Example 3.

Prescription example of nutrition cream in the cosmetic containing betulin of Example 1 of the present invention is as follows.

ingredient Content (% by weight) Betulin 0.5 Stearic acid 2.0 Cetyl alcohol 2.0 Glyceryl Monostearate 2.0 Polyoxyethylene sorbitan monostearate 0.5 Sorbitan sesquioleate 0.5 Glyceryl Monostearate / Glyceryl Stearate / Polyoxyethylene Stearate 1.0 Wax 1.0 Liquid paraffin 4.0 Squalane 4.0 Carlylic / Capric Triglycerides 4.0 Carboxy Vinyl Polymer 0.3 Butylene glycol 5.0 glycerin 3.0 Triethanolamine 0.5 Paraoxybenzoic acid propyl 0.2 Methyl paraoxybenzoate 0.2 Purified water Remaining amount system 100.0

Betulin according to the present invention is excellent in inhibiting collagenase activity, promoting collagen production and cell proliferation effect, and having excellent stability when blending, and thus improving wrinkles of the skin including the same, and also exhibiting excellent cell regeneration and collagen production promoting effect.

Claims (3)

Skin anti-wrinkle cosmetic composition comprising betulin. The skin wrinkle improvement cosmetic composition according to claim 1, wherein betulin is contained in an amount of 0.0001 to 3% by weight based on the total dry weight of the cosmetic composition. The skin wrinkle improvement cosmetic composition according to claim 1, which has a formulation of softening cream, converging cream, nutrient cream, nourishing cream, massage cream, essence and pack.

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