KR20070089459A - Composition for skin whitening containing mulberry root extract wrapped in cholesteryl hemisuccinate incorporated lipid-based nano-carriers - Google Patents

Abstract

A lipid-based nano-carrier is provided to increase the whitening effect of an extract of Mori Cotex Radicis by encapsulating an extract of Mori Cortex Radicis in the lipid-based nano-carrier in which cholesteryl hemisuccinate is introduced, thereby effectively inhibiting the melanogenesis without side effects. The lipid-based nano-carrier is characterized in that it is in the form of a liposome dispersion or a nano-emulsion in which cholesteryl hemisuccinate encapsulating an extract of Mori Cortex Radicis or a salt thereof is introduced. Regarding the total weight of the total liposome dispersion or the nano-emulsion, the content of the lipid is 0.001-20 wt.% and the content of the extract is 0.001-30 wt.%. The skin whitening composition comprises the nano-carrier.

Description

COMposition for skin whitening containing mulberry root extract wrapped in cholesteryl hemisuccinate incorporated lipid-based nano-carriers as cholesteryl hemisuccinate-based nanocarriers

1 is a graph showing the effect of the extract of baekbaekpi on the enzyme activity of glucosidase, one of the enzymes involved in the glycosylation process.

Figure 2 is a graph showing the effect of baekpipi extract on melanin production of HM3KO human melanoma cells. (C: control; A0.5 and A5: positive control group treated with arbutin 0.5 nm and 5 nm; phase 1, phase 10, phase 100, phase 200 and phase 500: 1 ppm, 10 ppm, 100 ppm, 200 ppm, and 500 ppm of lettuce extract )

Figure 3 is a protein electrophoresis picture showing the effect of baekryepi extract on the glycosylation of tyrosinase. (C: control; phase 200: 200 ppm of epidermis extract; A5: positive control group treated with arbutin 5 nm; Endo H: endoglycosidase H)

Figure 4 is a graph comparing the melanin production inhibitory effect of the extract of the lettuce extract collected on the nano-level lipid-based carrier in which the lettuce extract and cholesteryl hemisuccinate is introduced. (C: control group; W: liposome dispersion itself; W + phase 10 and W + phase 100: liposome dispersions collected to be 10 and 100 ppm of lettuce extract; phase 1, phase 10, phase 100, phase 200 and phase 500: 1 ppm, 10 ppm , 100 ppm, 200 ppm, and 500 ppm of lettuce extract)

The present invention relates to a skin whitening composition containing as an active ingredient the epidermis extract as a biologically active ingredient in a nano-level lipid-based carrier into which cholesteryl hemisuccinate is introduced. More specifically, the composition for skin whitening according to the present invention is an epidermis which is an active ingredient by lipid-based nanocarriers such as liposomes or nanoemulsions prepared using cholesteryl hemisuccinate or cholesteryl hemisuccinate salt. It relates to a skin whitening composition that can effectively inhibit melanin production without side effects by increasing the whitening effect of the extract.

Melanin, which determines human skin color, is made from skin cells called melanocytes and migrates to epidermal cells called keratinocytes. Here, melanin plays an important role such as forming a hat-like structure around the nucleus to protect genes from ultraviolet rays and to remove free radicals to protect intracellular proteins. There are no enzymes that break down melanin in vivo, but they are removed in such a way that keratinocytes fall off the skin as they fall off the epidermis. However, if the melanin is produced more than necessary, it causes a hyperpigmentation such as blemishes, freckles, spots, etc., which brings cosmetically bad results. In addition, as the leisure population increases, the number of people who enjoy working outside increases the need to prevent melanin pigmentation caused by ultraviolet rays. Therefore, it was necessary to develop a whitening agent that prevents excessive melanin production and many efforts have been made.

Until now, the development of whitening agents has been mainly focused on finding a substance that reduces melanin by inhibiting the activity of tyrosinase, an essential and most important enzyme in melanin biosynthesis. The whitening agent thus developed includes kojic acid, arbutin, glutathione, vitamin A, vitamin C, etc., but it does not have a satisfactory whitening effect for consumers and has a limited problem in its amount due to side effects. Therefore, it is urgent to develop a whitening agent that is more effective and safer than ever before.

On the other hand, tyrosinase, an enzyme that synthesizes melanin, is a glycoprotein, which is produced through glycosylation, a glycoprotein synthesis process in vivo. Many enzymes are involved in the glycosylation process, the most important of which is glucosidase. It has been found that the baekbaekpi extract already has a whitening effect has been filed a patent (Korean Patent Registration 1992-0002109, 1995-0016692) for its manufacturing method and efficacy. However, it is not known that the whitening efficacy of the extract of Morus alba was due to the mechanism of inhibiting glycosylation of tyrosinase. In particular, no specific studies have yet been mentioned on the enhancement of the whitening efficacy of the extracts of the epidermis by the nano-level lipid-based carrier (Korean Patent Application 2004-0117940) into which cholesteryl hemisuccinate is applied.

Accordingly, the present inventors have studied to provide a new whitening agent having excellent whitening effect and no side effects, the composition for skin whitening according to the present invention using cholesteryl hemisuccinate or cholesteryl hemisuccinate salt By confirming that the whitening effect of the extract of the epidermis, the active ingredient, is increased by the lipid-based nanocarriers such as liposomes or nanoemulsions, the present inventors have found that melanin production can be effectively suppressed without side effects, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a composition for skin whitening containing the extract of baekhyeolpi as an active ingredient, which is collected with cholesteryl hemisuccinate-based nanocarriers.

In order to achieve the above object, the composition for skin whitening according to the present invention provides a composition for skin whitening, characterized in that it contains an extract of baekryepi collected by cholesteryl hemisuccinate-based nano-carrier as an active ingredient.

Hereinafter, the present invention will be described in more detail.

Nanocarriers in which the extract of baekryepi as a physiologically active ingredient is incorporated into the skin whitening composition of the present invention is based on cholesteryl hemisuccinate and its salts.

The cholesteryl hemisuccinate (CHEMS) is a carboxyl group derived from a cholesterol terminal, the CHEMS alone; Or one selected from salts of CHEMS such as tris salt, tris (hydroxymethyl) aminomethane salt, cyclohexyl ammonium salt, and the like; Alternatively, a mixture of two or more selected from CHEMS and the salts of CHEMS described above may be used in combination with a lipid, which may be appropriately selected depending on the characteristics of the carrier or the manufacturing process.

Lipid-based nanocarriers according to the invention can be prepared in the form of liposomes or nanoemulsions. In preparing such liposomes or nanoemulsions, the lipid membrane structure can be stabilized by using the above-described CHEMS as a component of the lipid membrane structure. On the other hand, the lipid-based nanocarrier is sensitive to acidity, the lipid film stability is lowered in the region below the acidity of 6.0, and at a lower acidity, the decay is made to release the extract of baekryepi (physiologically active ingredient) embedded in the inner phase.

As a result, the substrate-based nanocarrier can be applied to the external preparation for skin whitening, which can maximize the whitening effect of the lettuce extract, since it can effectively release the lettuce extract embedded in the internal organ according to the acidity change. The preparation of the composition is possible.

In preparing liposomes or nanoemulsions of the present invention, phospholipids or nitrogenous lipids having fatty acid chains having 12 to 24 carbon atoms may be used as lipid components of lipid structures. Phospholipids are generally more preferred, for example, egg yolk lecithin (phosphatidylcholine), soybean lecithin, risorecitin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatitylethanolamine, diphosphatidyl Natural phospholipids such as glycerol, cardiolipin, and plasmagen; Hydrogenated products obtainable from these phospholipids by conventional methods; And disetylphosphate, distearoyl phosphatidylcholine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostaroylphosphatidylcholine, eleostaroylolamine, Synthetic lipids such as eleostaroylphosphatidylserine; Derivatives thereof; And fatty acid mixtures obtainable by their hydrolysis.

Lipids containing phospholipids may be used alone or in combination of two or more. When two kinds of phospholipids are mixed and used, Preferably, for example, the following combinations; Phosphatidylcholine: phosphatidylethanolamine, phosphatidylcholine: phosphatidylglycerol, phosphatidylcholine: phosphatidyl inositol, phosphatidylcholine: phosphatidinic acid, phosphatidylcholine: dioleoylphosphatidylethanolamine and the like. The mixing ratio is different depending on the component composition to be mixed, and the mixing ratio of the maximum component to the minimum component is preferably 1: 5 or less. For example, phosphatidylcholine: dioleoylphosphatidylethanolamine may be 1: 1 or 2: 1, 3: 1, 4: 1, 5: 1, 1: 1 in a molar ratio of 5: 1 to 1: 5, respectively. 5, 1: 4, 1: 3, 1: 2 and the like can be produced in various ways. In the case where three kinds of phospholipids are mixed and used, for example, phosphatidylcholine: dioleoylphosphatidylethanolamine: phosphatidylserine in a molar ratio range of 1: 5 or less, respectively, 1: 1: 1 or 2: 1 It can be produced in various mixing ratios such as: 1, 3: 1: 3, 3: 2: 1, 3: 2: 2, 4: 1: 1, 4: 2: 1.

As a method for forming a nanocarrier containing the extract of baekryepi by using the lipid component added CHEMS proposed in the present invention, after dissolving the phospholipids and CHEMS in an organic solvent, the solvent is evaporated, reduced pressure to form a lipid film Adding an aqueous solution and then applying ultrasonic waves; Dispersing phospholipids and CHEMS dissolved in an organic solvent in an aqueous solution and then applying ultrasonic waves; Dispersing or dissolving phospholipids and CHEMS in an organic solvent and then extracting or evaporating the organic solvent with excess water; Dispersing or dissolving phospholipids and CHEMS in an organic solvent, vigorously stirring the mixture using a homogenizer or a high pressure emulsifier and evaporating the solvent; Dispersing or dissolving phospholipids and CHEMS in an organic solvent and dialysis with excess water; Phospholipids and CHEMS are dispersed or dissolved in an organic solvent, and then water is gradually added.

In relation to the above production method, in order to dissolve phospholipids and CHEMS in an organic solvent, mechanical force may be applied or heated to 20 to 100 ° C, preferably 70 ° C or less. When using the salt of CHEMS, the salt of CHEMS can be dissolved and used in aqueous solution.

The nanocarrier of the present invention obtained by the above-described manufacturing method has an average particle diameter of 10 to 1,000 nm, more preferably 10 to 500 nm, and has a sensitive sensitivity to changes in acidity, depending on the composition and preparation method of the lipid membrane. It is possible to enhance the effect as a transporter by effectively releasing the lettuce extract incorporated in the cell.

Therefore, it can play a role as a transporter that can double the physiological activity of the baekbaekpi extract having a whitening effect as a physiologically active ingredient, the particle size is fine, colloidal stability is excellent and has excellent chemical stability, various skin external preparation formulation Can be used for In addition, the phospholipid construct has an advantage that is harmless to the human body can be usefully used in cosmetics for skin whitening.

Epidermis extract contained as an active ingredient in the composition for skin whitening of the present invention inhibits glycosylation of tyrosinase to prevent melanin production, thereby exhibiting a whitening effect. The cholesteryl hemisuccinate-based liposome dispersion may be used in an amount of 0.001 to 30% by weight, and preferably 0.2 to 20% by weight, according to the total weight of the skin extract composition, based on the total weight of the skin whitening composition. This is because the effect can not be expected at less than 0.001% by weight, there is a difficulty in the safety or formulation formulation above 30% by weight.

The composition for skin whitening according to the present invention is not particularly limited in its formulation, but specifically includes conventional cosmetic formulations such as lotion, nutrition cream, nutrition lotion, massage cream, nutrition essence and the like.

Hereinafter, although an Example and a test example are given and this invention is demonstrated in detail, this invention is not limited only to these examples.

Example 1 A method for preparing lettuce extract

50 g of lettuce was cut into pieces and 100 ml of 95% hydrous alcohol was added to the dry weight. Cooling condenser was installed to prevent evaporation of the active ingredient in a state of heating for 10 hours to 50 ~ 100 ℃ to extract the active ingredient, filtered and concentrated under reduced pressure, dried completely to obtain 3.5 g of baekpipi extract.

[Examples 2 and 3] A cholesteryl hemisuccinate-based liposome dispersion preparation method

11.7 mg of phosphatidylethanolamine or dioleoylphosphatidylethanolamine and 5 mg of cholesteryl hemisuccinate (CHEMS) are mixed with 1.25 ml chloroform: methanol (95: 5, vol / vol) After dissolving in a solvent, the solvent was evaporated under reduced pressure to form a film. To the film from which the organic solvent was removed, 1.25 ml of purified water obtained by dissolving 5 mg of the lettuce extract, the whitening efficacy component made in Example 1, was added to ultrasonic waves to prepare a liposome dispersion.

Liposome Preparation Conditions According to the Ratio of Phospholipids and Cholesterol Hemisuccinate (CHEMS) Phospholipids Phospholipids: CHEMS molar ratio Lipid component ratio (% by weight) in lipid dispersion Example 2 Phosphatidylethanolamine 3: 2 0.2 Example 3 Dioleoylphosphatidyl-ethanolamine 8: 2 0.2

Phospholipid types and ratios used in the above examples are not limited thereto, and the lipid components in Examples 2 to 3 were prepared by 0.2% of the total liposome dispersion, but the weight ratio is not limited thereto. In consideration of efficacy and safety and the like 0.001% to 20% by weight is possible, preferably 0.2% to 10% by weight.

[Examples 4 and 5 and Comparative Examples 1 and 2]

Using the liposome dispersions according to the present invention (Examples 2 and 3) according to the conventional preparation method, the composition for skin whitening of Examples 4 and 5 and Comparative Examples 1 and 2 as a cream formulation Prepared.

Liposomal Cream ingredient Example 4 Example 5 Comparative Example 1 Comparative Example 2 Glyceryl Stearate Polysorbate 60 Cetostearate Beeswax Squalane Solbitan Sesquioleate Cetylethylhexanoate Liquid Paraffin Glycerin Propylene Glycol Example 2 Example 3 Epidermis Extract Preservative Pigment Flavored Water 3.0 1.0 2.5 1.0 2.0 0.5 0.5 5.0 3.0 3.0 2.0--Trace Trace Trace to 100 3.0 1.0 2.5 1.0 2.0 0.5 0.5 5.0 3.0 3.0-2.0-Trace Trace Trace to 100 3.0 1.0 2.5 1.0 2.0 0.5 0.5 5.0 3.0 3.0---Trace Trace Trace to 100 3.0 1.0 2.5 1.0 2.0 0.5 0.5 5.0 3.0 3.0--2.0 Trace Trace Trace to 100

[Test Example 1]

In order to confirm the whitening effect of the compositions prepared in Examples 4 and 5 and Comparative Examples 1 and 2, an opaque tape having 6 holes of 1.5 cm diameter was attached to the upper forearm of the subject of 12 healthy men. Afterwards, the skin is irradiated by irradiating with ultraviolet light (UVB) 1.5 to 2 times the minimum erythema dose of each subject, and two months after applying the test substances, Contrast was measured. Each of the test materials was corrected twice daily (morning and evening) for the compositions of Examples 4 and 5 and Comparative Examples 1 and 2. The determination of the effect was determined by obtaining a "L" value representing the contrast of the skin (unburned Korean skin color generally represents a value of 50-70). When the test materials are applied, the L value is gradually increased, and the comparison between the test materials is a ΔL value, that is, a value obtained by subtracting the initial L value from the later L value (ΔL = final L value-application of the test substance. Previous value). The larger the ΔL value, the greater the whitening effect. The results are shown in the following [Table 3].

division Example 4 Example 5 Comparative Example 1 Comparative Example 2 DL 2.12 2.56 1.05 1.52

As shown in [Table 3], the ΔL values of the compositions of Examples 4 and 5 containing the extracts of lettuce extract collected with cholesteryl hemisuccinate-based nanocarriers as active ingredients were 2.12 and 2.56, respectively. The ΔL values of 1 and 2 were higher than 1.05 and 1.52, respectively.

[Test Example 2] Effect of Morus bark Extract on Enzyme Activity of Glucosidase

Glucosidase recognized the glycoside of p-nitrophenyl glycoside (PNP-glycoside) as a substrate and separated PNP and glycoside by enzymatic reaction, and the amount of PNP absorbed was absorbed at 405 nm. It measured and calculated | required. Enzymatic activity of glucosidase was indirectly determined through the relative amount of PNP compared to the control group. In a specific experimental method, glucosidase (0.38 u / ml) and an experimental sample were added to 50 mM sodium phosphate buffer (pH 6.8) and reacted at 37 ° C. for 1 hour. Nitrophenyl glycoside (0.2 mM) was added thereto and reacted at 37 ° C. for 5 to 10 minutes, and 0.3 M Na ₂ CO ₃ was added to measure absorbance at 405 nm. As a control, an enzyme reaction solution obtained by treating only a vehicle (EtOH: Propylene glycol: water = 3: 5: 2) in which a test sample was dissolved was used.

From the results of the following (Fig. 1), the extracts of the cedar extract were statistically significant (* P <0.01, vs. control) at 21, 42, 83, 167 and 333 ppm compared to the control (0 ppm) without the test sample added. It was confirmed that the enzyme activity of glucosidase inhibited by 36.56, 70.52, 82.90, 86.67 and 94.46%, respectively. Therefore, it was confirmed that the extract of E. coli effectively inhibited the effective activity of the glucosidase involved in glycosylation of the protein.

[Test Example 3] Determination of the effect of lettuce extract on the melanogenesis of human melanoma cells

HM3KO cells (Y. Funasaka, Department of dermatology, Kobe university school of medicine, 5-1 Kusunoki-cho 7-chrome, Chuo-ku, Kobe 650, Japan), human melanoma cells, have a 10% fetal bovine serum Tomycin (streptomycin) in 200 nM, penicillin (phenicillin) in a minimum Essential Medium (MEM) medium containing 100,000 U / L was incubated under 37 ℃, 5% CO ₂ conditions. The cultured cells were spread over 75 T flasks so that the number of cells was 3 × 10 ⁵ per flask, and the samples were added to the same medium and treated with the cells from the next day after waiting for the cells to adhere to the base wall overnight. Samples were treated by dissolving in vehicle (vehicle, ethanol: propylene glycol: water = 5: 3: 2) so that the extracts were 1, 10, 100, 200 and 500 ppm, respectively. In addition, the control group used a medium containing only the vehicle used to dissolve the sample, and arbutin (arbutin) as a positive control was used by dissolving in the vehicle so that 0.5, 5mM when treated in the medium. The fresh medium containing the sample was changed every 1 to 2 days and cultured until the cells were filled in the flask. When the cells were grown, the cells were collected and completely dissolved in 1N NaOH, and the absorbance at 490 nm was measured to determine the amount of melanin. When the amount of melanin in the control group is 100%, the relative melanin amount is calculated and expressed as a graph compared to the control group. The results are shown in the following (Fig. 2).

From the results of the following (Fig. 2), the total melanin amount divided by the protein amount was corrected as a result, arbutin was 21.82% and 5 (A5), 0.5 (A0.5) mM concentrations, respectively, compared to the control group was not added It was found that it has a melanin production inhibitory effect of 9.45%. The extracts of melancholia showed statistically significant (* P <0.01, vs. control) melanin production inhibitory effects of 57.93, 39.52 and 20.84% at concentrations of 500, 200 and 100 ppm, respectively. Therefore, it was confirmed that the extract of Morus vulgaris shows a whitening effect by inhibiting melanin production.

[Test Example 4] Determination of the effect of lettuce extract on glycosylation of tyrosinase in human melanoma cells

After HM3KO cells were grown in the same manner as in Test Example 3, the cells were collected and placed in a cell lysis solution (lysis buffer: 2% CHAPS in 50 mM Hepes and 200 mM NaCl, pH 7.5, protease inhibitors) and sonicated by ultrasound. In this case, 5 mM of arbutin and 200 ppm of lettuce extract were used as samples. The cell wash solution was centrifuged at 4 ° C. and 12,000 rpm for 10 minutes to separate and remove unbroken cells and melanin (melanin), and only the supernatant was taken. Endoglycosidase H (125 units) was added to the supernatant (protein amount: 20 mg), and the enzymes were reacted at 37 ° C. for 1 hour, and the proteins in the supernatant were separated by size under electrophoresis. Among the proteins isolated by electrophoresis, tyrosinase was identified by western blotting using an antibody specific for tyrosinase.

Tyrosinase, in which sugar residues are normally formed, appears as a glycoprotein of about 80 kD due to the enzymatic degradation of sugar residues by endoglycosidase H, and the enzymatic activity of glucosidase involved in glycosylation is inhibited. Tyrosinase, in which normal sugar residues are not formed, is hydrolyzed by endoglycosidase H, resulting in protein with a size of about 60 kD. The results are shown in the following (Fig. 3).

From the results of the following (Fig. 3), when the epithelium extract is treated with 200 ppm (200) of human melanoma cells, glycosylation of tyrosinase does not occur normally, and the sugar portion thereof is caused by endoglycosidase H (Endo H). Hydrolyzed (Tyrosinase + Endo H), and therefore, the amount of sugar-attached tyrosinase (about 80 kD) was confirmed to be reduced compared to the control (C). However, treatment with arbutin 5 mM (A5) did not change the amount of tyrosinase attached to the sugar. When Endo H was not treated, there was no change in the total tyrosinase expression level in all test substances. Therefore, it was determined that the extract of E. coli inhibited melanin production by inhibiting glycosylation of tyrosinase.

[Test Example 5] Determination of the effect of cholesteryl hemisuccinate-based epidermis extract on melanin production in human melanoma cells

In the same manner as in Test Example 3, the effect of inhibiting melanin production was compared between the extracts of the lettuce extract collected with the cholesteryl hemisuccinate-based nanocarrier. At this time, 1, 10 and 100 ppm of the extract of baekpipi as a sample, liposome dispersion liquid collected so that the extract of baekpipi at 10 and 100 ppm at the final test concentration in Example 2 was used. In addition, the control group was MEM medium (10% fetal calf serum, streptomycin) containing a vehicle (vehicle, ethanol: propylene glycol: water = 5: 3: 2) used to dissolve the test sample 200 nM, containing penicillin 100,000 U / L), and MEM medium (10% fetal calf serum, straptomycin) treated only with liposome dispersions without extracting the epidermis extract to measure the effect of liposome dispersion itself. (streptomycin) 200 nM, including penicillin 100,000 U / L) was also included in the test group.

From the results of the following (Fig. 4), as a result of correcting the total melanin amount divided by the protein amount, the extract of baekryepi skin at 10 (phase 10) and 100 (phase 100) ppm compared to the control group (C) to which the sample was not added Inhibition of melanin production was 17.18 and 25.93%, respectively, but the treatment of the same concentrations of the extracts of lilium extracts (W + phase 10, W + phase 100) to increase the melanin production inhibitory effect was 44.31 and 61.52%. Therefore, it was confirmed that the use of cholesteryl hemisuccinate-based nanocarrier enhances the melanin production inhibitory effect of the extract of lettuce.

As described above, the skin whitening composition according to the present invention contains the extract of the epidermis extract as a physiologically active ingredient in a nano-level lipid-based carrier to which cholesteryl hemisuccinate is introduced as an active ingredient without side effects. It can effectively suppress melanin production.

Claims (4)

Lipid-based nanocarriers in the form of liposome dispersions or nanoemulsions in which cholesteryl hemisuccinate or cholesteryl hemisuccinate salts, wherein the extracts of the epidermis were collected as bioactive ingredients. The nanocarrier according to claim 1, wherein the lipid component of the nanocarrier is 0.001 to 20% by weight based on the total amount of liposome dispersion or nanoemulsion. The method of claim 1, wherein the physiologically active active ingredient, baekbaekpi extract is characterized in that the nanocarrier containing 0.001 to 30% by weight relative to the total amount of liposome dispersion or nanoemulsion. A composition for skin whitening comprising the nanocarrier of claim 1.

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