KR102303202B1 - Multi-lamellar liposome with black rice bran derived material and method for preparing same - Google Patents

Abstract

The present invention relates to a multilamellar liposome containing a material derived from black rice bran, and provides a cosmetic composition having whitening, inflammation relief, and heavy metal adsorption effects using an active ingredient extracted from black rice bran. In addition, the present invention provides a cosmetic composition with improved formulation stability and improved skin permeability by encapsulating an active ingredient extracted from black rice bran in multilamellar liposomes.

Description

Multi-lamellar liposome with black rice bran derived material and method for preparing same

The present invention relates to multi-lamellar liposomes containing a material derived from black rice bran, and more particularly, to multi-lamellar liposomes capable of effectively capturing active ingredients contained in black rice bran.

Among the methods for extracting fat-soluble substances contained in plants, etc., the most commonly used method is the organic solvent extraction method. In the organic solvent extraction method, a plant is placed in a container, a solvent is added, the components contained in the plant are sufficiently dissolved, and the solvent is volatilized to obtain an extract. As the solvent, ethers, benzene, hexane, and other alcohols are used.

In particular, when manufacturing vegetable oil such as soybean oil, more edible oil can be obtained by using the hexane extraction method, so most companies are using the hexane extraction method. However, as it is known that hexane remains in the extract extracted by the hexane extraction method, concerns about the side effects of the hexane extraction method are growing.

Hexane (hexane, C ₆ H ₁₄ ) is an aliphatic hydrocarbon, manufactured from petroleum, and used as a raw material for strong adhesives, deodorants, inks, etc., or as a solvent for extracting fat-soluble substances contained in plants. However, when exposed to humans, there is a risk of respiratory system irritation, organ damage, skin irritation, eye irritation, etc.

If the hexane extraction method is used, it has the advantage of being able to mass-produce it at low cost, but research and development to solve the risk problem of residual hexane should be continuously performed.

Republic of Korea Patent No. 10-1560575 (2015.10.08.) describes a natural functional cosmetic composition containing black rice bran extract as an active ingredient. Republic of Korea Patent No. 10-1686490 (2016.12.08.) discloses a composition for preventing or treating bone metabolic diseases using black rice extract as an active ingredient.

The present invention proposes a new extraction method to solve the side effects of the hexane extraction method.

In addition, the present invention aims to provide a cosmetic composition with improved formulation stability and improved skin permeability by improving the formulation of a cosmetic composition using an active ingredient extracted from black rice bran.

The present invention includes a multilamellar liposome having a fat-soluble layer and a water-soluble layer as a multi-layer, wherein the fat-soluble layer contains olizanol (γ-oryzanol) and tocopherol, which are fat-soluble substances derived from black rice bran supercritical extract, It provides a cosmetic composition, characterized in that the water-soluble layer is a water-soluble substance derived from the black rice bran supercritical extract, phytic acid (phytic acid) is collected.

In the present invention, the cosmetic composition may be preferably for whitening.

In the present invention, the cosmetic composition may be one for alleviating skin inflammation, preferably or for alleviating nitric oxide (NO)-mediated inflammation.

In the present invention, the cosmetic composition may be preferably for heavy metal ion adsorption and removal.

In addition, the present invention supercritical extraction of black rice bran with carbon dioxide to obtain a supercritical extract of black rice (a); extracting olizanol (γ-oryzanol) and tocopherol from the black rice bran supercritical extract (b); (c) extracting phytic acid by acid hydrolysis in the black rice bran supercritical extract remaining after extracting the olizanol (γ-oryzanol) and tocopherol (tocopherol); (d) mixing the extracted olizanol and tocopherol with an organic solvent and then with soybean lecithin; After the step (d), the step (e) of adding the phytic acid extracted in the step (c); After the step (e), drying under reduced pressure to form a lipid film (f); and adding a water-soluble solvent to the resulting lipid membrane, and homogenizing (g); provides a method for producing a multilamellar liposome comprising.

In the present invention, the acid hydrolysis of step (c) is preferably treated in HCl with a concentration of 0.5 to 2N at 20 to 90° C. for 30 to 180 minutes.

In addition, the present invention provides a cosmetic composition comprising the multi-lamellar liposome prepared by the method for producing the multi-lamellar liposome.

In the present invention, the cosmetic composition may be preferably for whitening.

In the present invention, the cosmetic composition may be one for alleviating skin inflammation, preferably or for alleviating nitric oxide (NO)-mediated inflammation.

In the present invention, the cosmetic composition may be preferably for heavy metal ion adsorption and removal.

The present invention can reduce the risk of side effects due to the residual organic solvent by using the supercritical extraction method.

In addition, the present invention provides a cosmetic composition having a whitening effect using an active ingredient extracted from black rice bran.

In addition, the present invention provides a cosmetic composition for alleviating skin inflammation that relieves nitric oxide (NO)-mediated inflammation using an active ingredient extracted from black rice bran.

In addition, the present invention provides a cosmetic composition for adsorbing heavy metals using an active ingredient extracted from black rice bran.

In addition, the present invention provides a cosmetic composition with improved formulation stability and improved skin permeability by improving the formulation of a cosmetic composition using an active ingredient extracted from black rice bran.

1 is a graph showing the antioxidant activity ratio of black rice bran supercritical extract and black rice bran hexane extract.
2 is a graph showing the content of phytic acid according to the acid hydrolysis (HCl) concentration.
3 is a graph showing the total polyphenol content according to the acid hydrolysis (HCl) concentration.
4 is a graph showing the total flavonoid content according to the acid hydrolysis (HCl) concentration.
5 is a graph showing the content of phytic acid according to the acid hydrolysis temperature.
6 is a graph showing the total polyphenol content according to the acid hydrolysis temperature.
7 is a graph showing the total flavonoid content according to the acid hydrolysis temperature.
8 is a graph showing the content of phytic acid according to the acid hydrolysis time.
9 is a graph showing the total polyphenol content according to the acid hydrolysis time.
10 is a graph showing the total flavonoid content according to the acid hydrolysis time.
11 is a process diagram showing a method for preparing multi-lamellar liposomes using black rice bran supercritical extract and black rice bran supercritical extract foil (degreasing black rice bran).
12 is a field emission transmission electron microscope (FE-TEM) photograph of the multilamellar liposome containing the black rice bran-derived material of the present invention.
13 is a graph showing the formulation stability of the multi-lamellar liposome containing the black rice bran-derived material of the present invention.
14 is a graph showing the whitening efficacy of multilamellar liposomes containing black rice bran-derived substances.
15 is a graph showing the cytotoxicity of multilamellar liposomes containing black rice bran-derived material.
16 is a graph showing the NO production inhibitory ability of multilamellar liposomes containing black rice bran-derived substances.
17 is a graph showing the amount of heavy metal ions remaining after treatment with multilamellar liposomes containing black rice bran-derived substances.

The present invention includes a multilamellar liposome having a fat-soluble layer and a water-soluble layer as a multi-layer, wherein the fat-soluble layer contains olizanol (γ-oryzanol) and tocopherol, which are fat-soluble substances derived from black rice bran supercritical extract, It provides a cosmetic composition, characterized in that the water-soluble layer is a water-soluble substance derived from the black rice bran supercritical extract, phytic acid (phytic acid) is collected.

Black rice contains four times more anthocyanins than black soybeans, and is rich in anthocyanins, giving it a black color. Inorganic salts such as vitamins, iron, and zinc are contained 5 times more than normal rice, and it is known to effectively neutralize free radicals in the body as well as to prevent heart disease, adult disease, and cancer. Rice bran of black rice (black rice bran or black rice bran) contains various active ingredients such as olizanol, tocopherol, phytic acid, polyphenols, and flavoids.

Supercritical extraction is an extraction method using a supercritical fluid, also called supercritical fluid extraction. Carbon dioxide, water, ethane, ethylene, propane, etc. are used as the supercritical fluid. Among them, carbon dioxide has a low critical point (31.1℃, 73.8atm) and is non-toxic, so it is easy to extract and separate useful substances.

Black rice bran supercritical extract of the present invention contains fat-soluble components olizanol (γ-oryzanol) and tocopherol (tocopherol). On the other hand, black rice bran hexane extract was prepared using hexane as an extraction solvent and the antioxidant activity was measured. As a result, the antioxidant activity of the black rice bran supercritical extract of the present invention was 76.89%, which was higher than that of the black rice bran hexane extract 59.72%. .

On the other hand, the remaining foil after extracting the fat-soluble components from the black rice bran supercritical extract is called black rice bran supercritical extract foil (degreasing black rice bran). This is included. However, there is a problem in that it is difficult to obtain high-purity phytic acid because various impurities are mixed in the black rice bran supercritical extract foil. Accordingly, in the present invention, phytic acid was extracted by acid hydrolysis of black rice bran supercritical extract foil, and metal ions were removed through a strong ion exchange resin to obtain high-purity phytic acid.

The acid hydrolysis treatment is preferably performed at a concentration of 0.5 to 2N HCl at 20 to 90° C. for 30 to 180 minutes. Black rice bran supercritical extract In order to efficiently extract phytic acid, polyphenols, and flavonoids from foil (degreasing black rice bran), it is more preferable to extract with 2N HCl at 90° C. for 60 minutes.

In addition, the present invention supercritical extraction of black rice bran with carbon dioxide to obtain a supercritical extract of black rice (a); extracting olizanol (γ-oryzanol) and tocopherol from the black rice bran supercritical extract (b); (c) extracting phytic acid by acid hydrolysis in the black rice bran supercritical extract remaining after extracting the olizanol (γ-oryzanol) and tocopherol (tocopherol); (d) mixing the extracted olizanol and tocopherol with an organic solvent and then with soybean lecithin; After the step (d), the step (e) of adding the phytic acid extracted in the step (c); After the step (e), drying under reduced pressure to form a lipid film (f); and adding a water-soluble solvent to the resulting lipid membrane, and homogenizing (g); provides a method for producing a multilamellar liposome comprising.

The multi-lamellar liposome of the present invention is a multi-lamellar liposome, and refers to a lamellar vesicle in which a fat-soluble component and a water-soluble component exist in multiple layers.

It was confirmed that phytic acid was captured in the multi-lamellar liposome prepared according to the multi-lamellar reform manufacturing method of the present invention, and as a result of observation with a field emission transmission electron microscope (FE-TEM), spherical particles having a size of several tens of nanometers were confirmed.

In the present invention, the acid hydrolysis of step (c) is preferably treated in HCl with a concentration of 0.5 to 2N at 20 to 90° C. for 30 to 180 minutes.

In the present invention, the organic solvent of step (d) is, for example, anhydrous or hydrous lower alcohol having 1-4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, chloroform, butyl acetate, diethyl It may be an extractant selected from the group consisting of ether, dichloromethane, hexane, and mixtures thereof.

When the multilamellar liposome of the present invention was stored at 45° C. for 4 weeks, the phytic acid capture rate was 82.3% at week 0 and 81.0% at week 4, and it was confirmed that a stable formulation was maintained at 98.4% compared to the initial capture rate.

In addition, the present invention provides a cosmetic composition comprising the multi-lamellar liposome prepared by the method for producing the multi-lamellar liposome.

The cosmetic composition comprising the multilamellar liposome of the present invention showed tyrosine hydroxylase activity inhibitory ability, confirming that it had a whitening effect, and confirmed that it had an effect of alleviating nitric oxide (NO)-mediated inflammation.

In addition, it was confirmed that the skin permeability of phytic acid was excellent through the skin permeability test of the cosmetic composition comprising the multilamellar liposome of the present invention.

In addition, it was confirmed that the cosmetic composition comprising the multilamellar liposome of the present invention reduces the amount of heavy metal ions remaining in the skin by adsorbing heavy metal ions.

Hereinafter, the content of the present invention will be described in more detail in the following Examples and Experimental Examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of technical ideas equivalent thereto.

[Example 1: Obtaining olizanol and tocopherol through supercritical extraction from black rice bran]

Using carbon dioxide as a supercritical fluid, 100 g of black rice bran was subjected to a supercritical extractor at 50° C. and 400 bar to obtain a supercritical extract of black rice bran containing fat-soluble components olizanol (γ-oryzanol) and tocopherol (tocopherol).

[Comparative Example 1: Obtained olizanol and tocophenol through hexane extraction from black rice bran]

Add 1 L of n-hexane to 100 g of black rice bran, extract with shaking at room temperature for 4 hours, centrifuge the extract at 3,500 rpm for 10 minutes, and filter the supernatant with a Whatman filter (No. 1) and hexane with an evaporator. was removed and concentrated to obtain a hexane extract of black rice bran containing fat-soluble components olizanol (γ-oryzanol) and tocopherol (tocopherol).

[Experimental Example 1: Confirmation of antioxidant effect of black rice bran extract]

In this experiment, the antioxidant activity of the black rice bran extract obtained in Example 1 and Comparative Example 1 was measured.

A 0.2 mM DPPH (1,1-dipenyl-2-picrylhydrazyl) solution was prepared using ethanol, and 20 μl of the black rice bran extract of Example 1 and Comparative Example 1 was added to 180 μl DPPH solution, respectively, and mixed. After reacting at room temperature for 10 minutes, the absorbance was measured at 517 nm, and the absorbance ratio (%) was obtained to measure the antioxidant effect through DPPH scavenging ability.

As shown in FIG. 1 , the antioxidant activity of the black rice bran supercritical extract was 76.89%, which was higher than the antioxidant activity of 59.72% of the black rice bran hexane extract.

[Example 2: Extraction of phytic acid from black rice bran supercritical extract foil]

It was attempted to extract phytic acid from the black rice bran supercritical extract foil (degreasing black rice bran) from which the fat-soluble component of the black rice bran supercritical extract obtained in Example 1 was removed using hot water extraction and acid hydrolysis extraction.

Using hot water extraction as a control, 2L of purified water was added to 100 g of black rice bran supercritical extract foil (degreasing black rice bran), extracted at 80° C. for 1 hour, and the extract was centrifuged at 3,500 rpm for 10 minutes. Then, the supernatant was filtered through a Whatman filter ( No. 1) filtered. Metal ions were removed from the obtained extract through a strong ion exchange resin.

(1) Comparison of phytic acid content according to acid hydrolysis (HCl) concentration

0.5N, 1N, and 2N of HCl of 20 times was added to black rice bran supercritical extract foil, respectively, and extracted for 30 minutes at room temperature. After centrifugation of the extract at 3,500 rpm for 10 minutes, the supernatant was filtered with a Whatman filter (No. 1). filtered. Metal ions were removed from the obtained extract through a strong ion exchange resin.

(2) Comparison of phytic acid content according to acid hydrolysis temperature

20 times 2N HCl was added to black rice bran supercritical extract foil, extracted at room temperature, 40°C, 60°C, and 90°C for 30 minutes, respectively, and the extract was centrifuged at 3,500 rpm for 10 minutes. 1) was filtered. Metal ions were removed from the obtained extract through a strong ion exchange resin.

(3) Comparison of phytic acid content according to acid hydrolysis time

20 times 2N HCl was added to the black rice bran supercritical extract foil, extracted at 90°C for 30 minutes, 60 minutes, 120 minutes, and 180 minutes, respectively. After centrifuging the extract at 3,500 rpm for 10 minutes, the supernatant was filtered through Whatman filter (No. 1) was filtered. Metal ions were removed from the obtained extract through a strong ion exchange resin.

[Experimental Example 2: Measurement of phytic acid, polyphenol, and flavonoid contents of the extract obtained in Example 2]

In this experiment, the contents of phytic acid, polyphenol, and flavonoids contained in the black rice bran supercritical extract foil (degreasing black rice bran) obtained in Example 2 were measured.

The method for measuring phytic acid content is as follows. The sample was heated in boiling water for 5 minutes, cooled at room temperature, and centrifuged at 5,000 rpm for 10 minutes. The supernatant was taken and filtered through a membrane filter (0.45 μm) to be used as a test solution. Take 150 μl of the test solution, add 50 μl of wade reagent (0.03% ferric chloride and 0.3% sulfosalic acid), and mix for 5 seconds using a vortex mixer. The mixture was centrifuged at 3,000 rpm for 10 minutes, and then the supernatant was taken and absorbance was measured at 500 nm. For the preparation of the standard solution, a phytic acid solution having a concentration of 1,000 ppm was prepared and diluted with distilled water to prepare a standard solution for a calibration curve at a concentration of 1, 5, 10, 20, 50, 100 ppm.

The polyphenol content was measured by Folin-Denis method. After adding 10 μl of Folin-Ciocalteau phenol reagent and 100 μl of distilled water to 5 μl of the sample, the mixture was mixed at room temperature for 3 minutes. 100 μl of 20% Na ₂ CO ₃ solution was added to the mixture, and after reaction at room temperature for 1 hour, absorbance was measured at 725 nm using a spectrophotometer. Gallic acid was prepared in a concentration range of 25-800 μg/mL and analyzed in the same way as the sample, and the total phenol content of the sample was calculated from the standard calibration curve obtained.

The flavonoid content was measured by the method of Moreno et al. To 25 μl of the sample, 75 μl of ethanol, 140 μl of distilled water, 5 μl of 1M potassium acetate, and 5 μl of 10% aluminum nitrate were sequentially added and mixed, and after reaction at room temperature for 40 minutes, absorbance was measured at 415 nm. Using Quercetin (Sigma Co., USA) as a standard material, the total flavonoid content of the extract was calculated from a standard calibration curve obtained in a concentration range of 1 to 250 μg/mL.

The results of measuring the phytic acid, polyphenol, and flavonoid contents according to the HCl concentration by the above experimental method are shown in Tables 1 to 3 and FIGS. 2 to 10 below.

(1) Comparison of phytic acid content according to acid hydrolysis (HCl) concentration

menstruum Phytic acid (μg/g) Total polyphenols (mg/g) Total flavonoids (mg/g) hot water extract 404.31 12.19 4.30 0.5 N HCl 3549.27 16.19 4.42 1 N HCl 4079.57 17.04 4.46 2N HCl 4309.24 17.81 4.52

As shown in Table 1, the phytic acid content showed a tendency to increase as the concentration of HCl increased. In particular, in the case of acid hydrolysis with 2N HCl, 4309.24 μg/g of phytic acid was contained, and it was confirmed that the phytic acid content was higher than that of 404.31 μg/g in the case of hot water extraction (FIG. 2).

The content of total polyphenols showed a tendency to increase as the HCl concentration increased. In particular, it was confirmed that 17.81 mg/g of polyphenol was contained in the case of acid hydrolysis with 2N HCl, which increased by 5.62 mg/g compared to 12.19 mg/g in the case of hot water extraction (FIG. 3).

Total flavonoid content showed a tendency to increase with increasing HCl concentration. In particular, in the case of acid hydrolysis with 2N HCl, 4.52 mg/g of flavonoids were contained, and it was confirmed that it was increased by 0.22 mg/g compared to 4.30 mg/g in the case of hot water extraction (FIG. 4).

(2) Comparison of phytic acid content according to acid hydrolysis temperature

Temperature Phytic acid (μg/g) Total polyphenols (mg/g) Total flavonoids (mg/g) room temperature 4106.94 17.04 4.46 40℃ 4234.14 18.85 4.52 60℃ 4315.62 20.81 4.67 90℃ 4406.25 22.65 4.78

As shown in Table 2, the contents of phytic acid (FIG. 5), total polyphenols (FIG. 6), and total flavonoids (FIG. 7) tended to increase as the acid hydrolysis temperature increased. In particular, in the case of acid hydrolysis at 90°C, the highest contents were 4406.25 μg/g of phytic acid, 22.65 mg/g of total polyphenols, and 4.78 mg/g of total flavonoids.

(3) Comparison of phytic acid content according to acid hydrolysis time

hour Phytic acid (μg/g) Total polyphenols (mg/g) Total flavonoids (mg/g) 30 min 4354.58 17.00 4.50 60 min 4575.32 22.19 4.76 120 min 4245.61 20.27 4.67 180 min 4057.94 18.54 4.65

As shown in Table 3, the phytic acid content was the highest at 4354.58 μg/g after 60 minutes of acid hydrolysis (FIG. 8), and the total polyphenol content was 22.19 mg/g after 60 minutes of acid hydrolysis. It showed a high content (FIG. 9), and the total flavonoid content was also the highest at 4.76 mg/g when acid hydrolyzed for 60 minutes (FIG. 10).

Accordingly, it was confirmed that the acid hydrolysis conditions for efficiently extracting phytic acid, polyphenols, and flavonoids from the black rice bran supercritical extract (degreasing black rice bran) extract were preferably extracted with 2N HCl at 90° C. for 60 minutes.

[Example 3: Preparation of multilamellar liposomes containing material derived from black rice bran]

The Bangham method (BM) is a method of dissolving lecithin in an organic solvent containing a fat-soluble component, stirring, and drying under reduced pressure conditions with a reduced pressure concentrator to form a lipid film inside the flask. In the present invention, multilamellar liposomes were prepared by forming a lipid membrane using the Bangham method, then hydrating it with distilled water or a buffer solution in which the water-soluble component was dissolved, and then homogenizing it.

Phospholipids used to prepare liposomes were lecithin from which unsaturated components were removed by hydrogenation of lipids extracted from soybean, and Emulmetik 950 (Lucas Meyer) containing 95% or more of phosphatidyl choline was used. After mixing phospholipid and sodium deoxycholate in a ratio of 9: 1, the same amount of ethanol and fat-soluble extract (black rice bran supercritical extract of Example 1) was added and completely dissolved in a thermostat at 60° C. to prepare a transparent sol solution. After hardening it at room temperature, bring it back to a 60 ℃ thermostat, and when it becomes a transparent sol solution, a water-soluble extract (extracting the black rice bran supercritical extract foil (degreasing black rice bran) of Example 2 with 2N HCl at 90 ℃ for 60 minutes) is added. It was magnetically stirred for more than 10 min. After drying completely so that a lipid film is formed inside the flask under reduced pressure at 50 ° C with a vacuum concentrator, distilled water is added to the lipid film to hydrate, and homogenizing at 2000 rpm for 10 min. Liposomes were completed. The method for preparing multilamellar liposomes of the present invention is briefly shown in FIG. 11 .

In order to measure the phytic acid capture efficiency of the multilamellar liposomes prepared according to the above method, a certain amount of the liposome suspension was taken and the liposomes were removed using a 0.1 μm syringe filter. The collection efficiency was confirmed by measuring the content of phytic acid not captured in the liposome and the content of phytic acid in the initial extract. The collection efficiency was calculated according to the following Equation 1 for the content value of phytic acid.

[Equation 1]

The phytic acid content of the initial extract was 342.1 μg/ml, the content of phytic acid not captured in the liposome was 60.41 μg/ml, and the collection efficiency was calculated to be 82.34%.

As a result of observing the size and shape of the multilamellar liposomes prepared in this Example with a field emission transmission electron microscope (FE-TEM), as shown in FIG. 12 , spherical particles having a size of several tens of nanometers were confirmed.

[Experimental Example 3: Evaluation of the stability of multilamellar liposomes containing black rice bran-derived substances]

In this experiment, the formulation stability of the multilamellar liposome containing the black rice bran-derived material prepared in Example 3 was evaluated.

The multi-lamellar liposomes and black rice bran extract prepared in Example 3 (extracted from the black rice bran supercritical extract foil (degreasing black rice bran) of Example 2 with 2N HCl at 90° C. for 60 minutes) were shielded from light in a storage tube, respectively. It was stored at 45°C and stability was confirmed for 4 weeks. After removing the liposome using a 0.1 μm syringe filter, the content of phytic acid present in the filtrate was measured and compared with the change in the phytic acid content of the black rice bran extract. The phytic acid content was measured in the same manner as in Experimental Example 2.

As shown in Figure 13, the phytic acid content of the black rice bran extract showed a tendency to gradually decrease as time elapsed, whereas in the case of multilamellar liposomes, the phytic acid capture rate was 82.3% at week 0 and 81.0% at week 4, It was confirmed that the stable formulation was maintained at 98.4% compared to the initial collection rate.

[Experimental Example 4: Evaluation of whitening efficacy of multilamellar liposomes containing black rice bran-derived substances]

In this experiment, the whitening efficacy of the multilamellar liposome containing the black rice bran-derived material prepared in Example 3 was evaluated.

The inhibitory ability of tyrosine hydroxylase activity of tyrosinase, which is converted to 3,4-dihydroxyphenylalanine (DOPA) by hydroxylation of tyrosine, was evaluated. Multilamellar liposomes containing black rice bran extract were diluted in 0.1 M phosphate buffer (pH 7.0) to prepare test solutions of 0.5%, 1%, 2% and 5% concentrations, respectively. In a 96-well plate, 170 μl of 0.1 M phosphate buffer (pH 7.0), 10 μl of test solution diluted to various concentrations, 10 μl of 2000 unit/mL mushroom tyrosinase, and 10 μl of 10 mM L-DOPA were sequentially added and mixed at 37°C. After reacting for 15 minutes, absorbance was measured at a wavelength of 475 nm. The absorbance of the blank test solution was measured by adding 0.1 M phosphate buffer (pH 7.0) instead of the test solution and reacting in the same way. The enzyme activity inhibition rate was calculated according to Equation 2 below.

[Equation 2]

In order to investigate the effect of liposomes on the whitening efficacy, the activity inhibiting ability of tyrosinase was measured. 14, ascorbic acid 100 μM concentration of ascorbic acid inhibited tyrosine hydroxylase activity, which was a positive control, 75.6%, and liposome 1% sample showed a similar level of efficacy, 74.5%. increase was confirmed.

[Experimental Example 5: Evaluation of cell viability and NO production inhibitory ability of multilamellar liposomes containing black rice bran-derived material]

In this experiment, the cell viability and NO production inhibitory ability of the multilamellar liposome containing the black rice bran-derived material prepared in Example 3 were evaluated.

(1) Measurement of cell viability

RAW 264.7 cells, which are mouse macrophages, were purchased from the Korea Cell Line Bank and used. The viability of RAW 264.7 cells was measured by performing the MTT Assay (Denizot F and Lang R. J Immunological Method 89: 271-277, 1986). RAW 264.7 cells were aliquoted into a 96-well plate to 1 × 10 ⁵ cells/well and cultured for 24 hours. After culturing the cells for 24 hours, the cell culture medium was exchanged with a culture medium containing the liposome formulation at a concentration of 0.5%, 1%, 2%, 3%, 4%, 5% and 10%. After culturing the cells for 24 hours, the culture medium After removal, 1 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Amresco) solution was added, followed by incubation for 2 hours. After removing the MTT solution, MTT was reduced by mitochondrial dehydrogenase and the resulting blue formazan was dissolved by adding isopropanol, and then the absorbance was measured at 570 nm.

As shown in FIG. 15 , the liposome showed cytotoxicity at a concentration of 10% and a cell viability of over 90% was confirmed at a concentration of 5% or less. Below, the NO production inhibitory ability was evaluated at a concentration of 5% or less.

(2) NO production inhibitory ability evaluation

The NO concentration in the culture medium was measured using the griess reagent system, and RAW 264.7 cells were aliquoted in a 96-well plate at 1.5×10 ⁵ cells/well and cultured for 24 hours. After culturing, the liposome formulation was treated with 0.5%, 1%, 2%, 3%, 4% and 5% concentrations after replacement with a new culture medium, and LPS was treated with a concentration of 1 μg/ml and cultured again for 24 h. 50 μl of N1 buffer in 100 μl of culture medium was treated in each well and reacted at room temperature for 10 min. Then, 50 μl of N2 buffer was treated in each well and reacted for 10 min. Absorbance was measured at 540 nm. The concentration of NO in the culture medium was determined using a standard curve for each concentration.

As shown in FIG. 16, it was confirmed that NO production was reduced compared to the LPS-treated group, and as the concentration of liposomes increased, the tendency to decrease NO production was confirmed.

[Experimental Example 6: Confirmation of skin permeability of multilamellar liposomes containing black rice bran-derived material]

In this experiment, the skin permeability of the multilamellar liposome containing the black rice bran-derived material prepared in Example 3 was evaluated.

The skin permeability test method was performed by the skin pampa test. A blank was prepared with a prisma buffer and analyzed (reading). In the process of preparing a sample in a deep well plate, add 1,000 μl of prisma buffer to the deep well plate according to the pH-map, open the lid of the stock plate, and use an 8-channel pipette to remove the sample. 5 μl was taken and transferred to a deep well plate. After that, the pipette was adjusted to 500 μl and mixed 4 to 6 times, and the concentration of the solution was measured or calculated, and the result was used as a <reference> of the essay.

As a process of preparing a donor plate for a skin test assay, 200 μl of a sample solution from a deep well plate was transferred to the donor plate, and the lid was covered. At this time, the top plate of the PAMPA sandwich is an acceptor plate, and the bottom plate of the PAMPA sandwich is a donor plate.

In the process of preparing an acceptor plate and a PAMPA sandwich, an 8-channel pipette is adjusted to 200 μl, wells are hydrated, and an assay of a donor plate is prepared, The top plate was transferred from the hydration solution reservoir to the support plate. Then, 200 μl of prisma buffer of pH 7.4 was added to each well of the acceptor plate, and after removing the lid of the donor plate, be careful not to create bubbles. while slowly placing it on the donor plate from the bottom row (H) of the acceptor plate. At this time, after the sandwich is made, a lid is placed on the acceptor plate, and in order to prevent the skin membrane from drying out, it should be finished within 4 to 5 minutes and the experiment should be started. And place the sandwich in a humidity chamber or gut-box with a wet sponge to maintain high relative humidity to minimize evaporation, but never tilt the sandwich plate. And it was incubated without stirring for 5 hr at RT.

Next, as a process of donor/acceptor analysis (reading), the incubation is finished, the PAMPA sandwich is taken out from the humidity chamber, and the lid is opened 8 Using a channel pipettor, 150 μl of each was transferred from an acceptor plate to a UV plate, and then set to a range of 250 to 498 nm in a spectrophotometer and analyzed (reading).

To check the skin permeability of the multilamellar liposomes and black rice bran extract prepared in Example 3 (extracting the black rice bran supercritical extract foil of Example 2 (degreasing black rice bran) with 2N HCl at 90° C. for 60 minutes) The skin pampa system was used. The permeation rate (%) is shown in Table 4 below by comparing the phytic acid content of the black rice bran extract before permeation and the phytic acid content of the black rice bran extract and multilamellar liposome after permeation.

Phytic acid (μg/ml) Transmittance (%) Black rice bran extract before permeation 342.1 Black rice bran extract after penetration 6.64 1.94 Multilamellar Liposomes After Permeation 10.19 2.98

As shown in Table 4, the transmittance of the multilamellar liposome was 2.98%, confirming that the transmittance was increased by 1.04% compared to the transmittance of 1.94% of the black rice bran extract.

[Experimental Example 7: Evaluation of heavy metal adsorption efficacy of multilamellar liposomes containing black rice bran-derived substances]

In this experiment, in order to evaluate the heavy metal adsorption effect of the multilamellar liposome prepared in Example 3, residual heavy metals were measured through a skin absorption experiment using pig skin.

Purchase pig skin from which subcutaneous fat has been refrigerated and distributed on the same day ^{, cut it to a certain size so that the width and length are 5 × 5 cm 2} , and then evenly apply 1 ml of a heavy metal standard solution on the skin surface and dry it at room temperature for 5 hours. . The heavy metal standard solution was prepared using Pb, Cd, Zn, and As so that the heavy metal ion concentration was 1,000 ppm. After evenly applying 1 ml of 0.5%, 1%, 2%, 3%, 4%, and 5% concentration of multilamellar liposomes to the dried pig skin, 2 hours later, the skin surface was washed thoroughly with running water, wiped dry, and dried. To separate the epithelial skin, the epithelium was separated using tweezers after reaction at 60° C. for 1-2 minutes. Heavy metal ions remaining in the epithelium were analyzed by ICM/MS to quantify the amount of heavy metals.

As shown in FIG. 17, it was confirmed that the amount of heavy metal ions decreased compared to the control group not treated with the multi-lamellar liposome of the present invention. When the concentration of the multi-lamellar liposome of the present invention was 0.5%, the residual heavy metal ion was 22.4%, and when the concentration of the multi-lamellar liposome was 5%, it was 10.7%. As the concentration of the multi-lamellar liposome of the present invention increases, the amount of residual heavy metal ions A decrease was confirmed.

[Preparation Example 1: Cosmetic composition comprising multilamellar liposomes containing black rice bran-derived material]

A toner formulation was prepared with the cosmetic composition comprising the multilamellar liposome prepared in Example 3.

With reference to Table 5 below, phase A was weighed in a beaker and dissolved for 20 minutes with an azimuth mixer, and phase B was completely dissolved by heating phase B to 50° C. in another beaker, then phase B was cooled to room temperature, and then phase C was added. After mixing phase B and phase C, it was added to phase A and mixed for 10 minutes with an azimuth mixer.

Raw material name content(%) A
Purified water 70.87 64.87 66.33 70.87 72.79 Black rice bran-containing liposomes 5.00 5.00 5.00 5.00 5.00 green tea extract 4.00 4.00 5.00 4.00 4.00 Centella asiatica extract 3.00 3.00 2.00 3.00 3.00 propolis extract 3.00 10.00 5.00 3.00 3.00 glycerin 1.00 5.00 5.00 1.00 1.00 Sodium PCA 1.00 1.00 0.50 1.00 0.05 sodium lactate 1.00 1.00 0.00 1.00 0.05 Aloe Vera Leaf Extract 0.01 0.01 0.00 0.01 0.01 Sodium Phytate 0.03 0.03 0.00 0.03 0.03 B black rice bran extract 10.00 10.00 10.00 10.00 10.00 Polyquaternium-51 0.01 0.01 0.05 0.01 0.01 C hyaluronic acid 0.01 0.01 0.05 0.01 0.01 panthenol 0.01 0.01 0.01 0.01 0.01 1,2-Hexanediol 1.00 1.00 1.00 1.00 1.00 Ethylhexylglycerin 0.02 0.02 0.02 0.02 0.02 Citric Acid 0.04 0.04 0.04 0.04 0.02 Sum 100.00 100.00 100.00 100.00 100.00 Preparation 1-1 Preparation 1-2 Preparation 1-3 Preparation Example 1-4 Preparation 1-5

As a result of the feeling test of the cosmetic composition prepared according to the above method, the moisturizing power of Preparation Example 1-4 was the best, and when the toner of Preparation Example 1-4 was wetted on a cotton pad after cleansing and the skin was wiped off, the effect of removing the residue was reduced. was the best.

[Preparation Example 2: Cosmetic composition comprising multilamellar liposomes containing black rice bran-derived material]

A cleansing foam formulation was prepared with the cosmetic composition comprising the multilamellar liposome prepared in Example 3.

With reference to Table 6 below, after weighing phase B in a beaker and dissolving it with an azimuth mixer for 20 minutes, weighing the raw material of phase A on phase B, heating it at 80° C. for 1 hour to dissolve it completely, cooling it to room temperature, and then adding the raw material of phase C It was added and dissolved slowly.

Raw material name content(%) A
Purified water 27.80 37.80 27.80 32.80 27.80 Lauryl Glucoside 10.00 5.00 10.00 10.00 20.00 cocoglucoside 20.00 10.00 20.00 10.00 10.00 Decylglucoside 10.00 15.00 10.00 10.00 10.00 sodium
Cocoamphoacetate 10.00 10.00 10.00 15.00 10.00 glycerin 2.00 2.00 2.00 2.00 2.00 honey 1.00 1.00 1.00 1.00 1.00 Calendula Flower Extract 1.00 1.00 1.00 1.00 1.00 Glyceryl Caprylate 0.50 0.50 0.50 0.50 0.50 B black rice bran extract 10.00 10.00 10.00 10.00 10.00 Cellulose Gum 0.50 0.50 0.50 0.50 0.50 C Citric Acid 1.10 1.10 1.10 1.10 1.10 Jojoba Seed Oil 0.60 0.60 0.60 0.60 0.60 orange oil 0.50 0.50 0.50 0.00 0.00 lavender oil 0.00 0.00 0.00 0.50 0.00 eucalyptus oil 0.00 0.00 0.00 0.00 0.50 Black rice bran-containing liposomes 5.00 5.00 5.00 5.00 5.00 Sum 100.00 100.00 100.00 100.00 100.00 Preparation 2-1 Preparation Example 2-2 Preparation 2-3 Preparation 2-4 Preparation Example 2-5

As a result of a feeling test of the cosmetic composition prepared according to the above method, the foaming effect of Preparation Example 2-3 was the best, and showed a soft feeling. In addition, the cleansing power and skin moisturizing power of the cleansing foam were the best.

Claims (10)

Including a multilamellar liposome having a fat-soluble layer and a water-soluble layer as a multi-layer,
In the fat-soluble layer, olizanol (γ-oryzanol) and tocopherol, which are fat-soluble substances derived from the black rice bran supercritical extract, are collected in the oil-soluble layer, and phytic acid, a water-soluble substance derived from the black rice bran supercritical extract, is collected in the water-soluble layer. ) is collected,
The multi-lamellar liposome has increased phytic acid capture efficiency,
The phytic acid is extracted by acid hydrolysis of the black rice bran supercritical extract remaining after extracting the fat-soluble component from the black rice bran supercritical extract,
The acid hydrolysis, a cosmetic composition, characterized in that the treatment in HCl of 1 ~ 2N concentration at 60 ~ 90 ℃ for 30 ~ 60 minutes.
According to claim 1,
The cosmetic composition,
A cosmetic composition for whitening.
According to claim 1,
The cosmetic composition,
A cosmetic composition for relieving skin inflammation that relieves nitric oxide (NO)-mediated inflammation.
According to claim 1,
The cosmetic composition,
A cosmetic composition, characterized in that it is for removal of heavy metal ions by adsorption.
Supercritical extraction of black rice bran with carbon dioxide to obtain a supercritical extract of black rice bran (a);
extracting olizanol (γ-oryzanol) and tocopherol from the black rice bran supercritical extract (b);
(c) extracting phytic acid by acid hydrolysis in the black rice bran supercritical extract remaining after extracting the olizanol (γ-oryzanol) and tocopherol (tocopherol);
(d) mixing the extracted olizanol and tocopherol with an organic solvent and then with soybean lecithin;
After the step (d), the step (e) of adding the phytic acid extracted in the step (c);
After the step (e), drying under reduced pressure to form a lipid film (f); and
To prepare a multilamellar liposome, including adding a water-soluble solvent to the produced lipid membrane, and homogenizing (g);
The acid hydrolysis of step (c) is to be treated at 60 to 90 ° C for 30 to 60 minutes in HCl of 1 to 2N concentration,
The multilamellar liposome is provided with a multi-layered oil-soluble layer and a water-soluble layer, and the oil-soluble layer contains olizanol (γ-oryzanol) and tocopherol, which are fat-soluble substances derived from black rice bran supercritical extract, and the water-soluble layer is collected. In the layer, phytic acid, a water-soluble substance derived from the black rice bran supercritical extract, is collected, and the method for producing multilamellar liposomes, characterized in that the collection efficiency of the phytic acid is increased.
delete A cosmetic composition comprising the multilamellar liposome prepared by the method of claim 5 .
8. The method of claim 7,
The cosmetic composition,
A cosmetic composition for whitening.
8. The method of claim 7,
The cosmetic composition,
A cosmetic composition for relieving skin inflammation that relieves nitric oxide (NO)-mediated inflammation.
8. The method of claim 7,
The cosmetic composition,
A cosmetic composition, characterized in that it is for removal of heavy metal ions by adsorption.

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