KR102310044B1 - Cosmetic composition comprising lutein nanoencapsulated with cubosome as an active ingredient - Google Patents

Abstract

The present invention proposes nanoparticles that easily penetrate the skin by encapsulating lutein derived from PB17 (Auxenochlorella protothecoides, Chlorella minutissima ) that is cultured by the PSP method and extracted in a supercritical manner. PB17-lutein encapsulated nanoparticles are cubosomes made of monoolein and made into cubic phases. The size of cubosomes containing PB17-lutein is approximately 100-200 nanometers, and exhibits 30-40% transmittance in the artificial skin membrane. PB17-Lutein encapsulated in cubosome formed of monoolein presented in the present invention has excellent skin penetration, no skin irritation, and excellent antioxidant, whitening and wrinkle improvement effects.

Description

Cosmetic composition comprising lutein nanoencapsulated with cubosome as an active ingredient

The present invention relates to a cosmetic composition comprising, as an active ingredient, microalgae-derived lutein nanoencapsulated with cubosomes.

The application of natural molecules by nanotechnology systems to the skin can yield numerous clinical benefits in dermatology and cosmetics. In addition, several studies have demonstrated that glyceryl monoolein-based system derivatives are suitable for skin treatment. Encapsulation is a very useful technique to prevent undesirable changes resulting from harsh environmental conditions and unstable material properties. Through this method, the active compound is protected within the coating material, which improves stability and bioavailability. Capsules contain liquid crystal aggregates (liposomes and cubosomes) or crosslinked gel networks (hydrogels) that load, stabilize and finally deliver active ingredients to obtain a protective effect. These characteristics have led to the use of various encapsulation methods in the food and pharmaceutical industries.

A cubosome is a small biological capsule that can deliver nutrients or drug molecules with high efficiency. A cubosome has a very symmetrical internal structure in the form of a small hexagon, in which lipid molecules similar to those constituting a cell membrane are aggregated. This property is of great interest in the pharmaceutical and food fields because the structure of the cubosome can be used to improve the delivery of nutrients and drugs and to control the release of molecules. The most popular lipids used in cubosomes is monoolein (MO, 1-Oleoyl-rac -glycerol, C 21 H 40 O 4; synonyms: 1- (cis -9-Octadecenoyl) - rac -glycerol, rac -Glycerol 1-monooleate, DL-α-monoolein , Glyceryl cis -9-octadecenoate, MG (18 : 1(9Z)/0 : 0/0 : 0) [rac]) and its structural formula is as follows.

Monoolein is insoluble in water and cold alcohol, but very soluble in oil, petroleum ether, chloroform and even hot alcohol. In particular, monoolein is considered a highly desirable material as a food emulsion because it is a much more non-toxic, biodegradable and biocompatible material due to its high oil solubility. Monoolein is affected by lipolysis due to different kinds of esterase activity in different tissues. Cubosomes have the ability to encapsulate hydrophobic, hydrophilic, and amphiphilic substances. Cubosomes can increase the solubility of poorly soluble substances. The cubosome dispersion has excellent bioadhesiveness and biocompatibility. Because of its characteristics, cubosomes are a system that can be applied to the human body in various ways, such as oral, transdermal, and parenteral.

Lutein (C ₄₀ H ₅₆ O ₂ , Synonym: α-Carotene-3,3′-diol, β, ε-carotene-3,3′-diol, β, ε-carotene, Xanthophyll) is a type of plant pigment called carotenoid. and its chemical structure is as follows.

Since lutein is not formed by the body itself, it must be supplied through food or health supplements. Hundreds of carotenoids exist in nature, and lutein is one of 20 carotenoids present in the human body. Lutein is concentrated in the macular lutea, a small area of the human eye. Lutein plays an important role in improving vision and protecting the eyes from harmful short-wavelength blue light. Serum levels of lutein have also been shown to be inversely related to the risk of ophthalmic diseases such as macular degeneration and cataract formation. Lutein also plays an important role in the prevention of cardiovascular disease, stroke and lung cancer.

PCT/US2010/049347

It is an object of the present invention to present a functional cosmetic composition with improved efficacy by nano-encapsulating lutein, which is highly useful as a cosmetic composition for antioxidant, whitening, and wrinkle improvement, using cubosome.

In order to achieve the above object, the present invention provides a nanoencapsulated composition comprising lutein extracted from deposit strain PB17 as an active ingredient and using monoolein cubosome.

The deposit strain PB17 used in the present invention belongs to Chlorella minutissima taxonomically and contains a large amount of lutein.

In the present invention, the PB17 extract may be all extracts, fractions, purified products, dilutions, concentrates, or dried products obtained in each step of extraction, fractionation, or purification. In addition, the extract is extracted by water, alcohol, nucleic acid, or a mixed solvent thereof including purified water in addition to supercritical extraction, wherein the alcohol is a C1 to C4 lower alcohol, and the lower alcohol is ethanol, methanol or butanol, and the ethanol is characterized in that 10% to 99% of ethanol.

In the present invention, “PBEN17” refers to nanocapsules containing lutein extracted from PB17. The nanocapsule may be a cubosome composed of monoolein.

In the present invention, the term "active ingredient" means a component that can exhibit a desired activity alone or can exhibit activity together with a carrier that has no activity by itself.

In the present invention, the "active ingredient" was nanoencapsulated using cubosomes containing monoolein as a main component. The nanoencapsulated “active ingredient” permeated the synthetic membrane structure significantly and efficiently in a specific implementation of the present invention.

In another specific implementation, the nanoencapsulated material PBEN17 exhibited significant radical scavenging activity compared to the control, inhibiting xanthine oxidase, inhibiting tyrosinase activity, inhibiting collagenase activity, It was confirmed that the elastase activity was inhibited.

Therefore, the nanoencapsulated PB17 extract of the present invention can be very usefully used as a cosmetic composition through radical scavenging and inhibition of xanthine oxidase, tyrositase, collagenase, and elastase activity.

In order to achieve the object of the present invention, an embodiment of the present invention provides nano-sized cubosome crystals including lutein extracted from microalgae.

The microalgae may be characterized in that the accession number KCTC 18635P.

The cubosome may be characterized in that it is composed of monoolein.

The cubosome may be characterized in that it contains lutein (PB17 culture extract) at a concentration of 100 to 500 μg/ml, and monoolein dispersion at a concentration of 0.1 to 0.5 mg/ml.

In another embodiment of the present invention

preparing a lutein dispersion;

preparing a monoolein dispersion;

mixing the lutein dispersion and the monoolein dispersion;

filtering the mixed solution through a fine separation network;

When a precipitate is formed in the mixed solution, a method for producing cubosome nanoparticle crystals comprising the step of centrifuging is provided.

The lutein may be characterized in that it is extracted from microalgae of accession number KCTC 18635P.

The fine separation network may be a filter having a size of 0.1 to 0.5 micrometers.

In another embodiment of the present invention, a cosmetic composition comprising nano-sized cubosome crystals including lutein extracted from microalgae as an active ingredient is provided.

The microalgae may be characterized in that the accession number KCTC 18635P.

The cubosome may be characterized in that it is composed of monoolein.

The cubosome may contain lutein (PB17 culture extract) at a concentration of 100 to 500 μg/ml, and monoolein dispersion at a concentration of 0.1 to 0.5 mg/ml.

The cosmetic composition may be characterized in that it is for antioxidant, whitening, or wrinkle improvement.

As can be seen from the following experimental examples, the composition according to the present invention is a cosmetic composition and has excellent effects of radical scavenging and inhibiting xanthine oxidase, tyrositase, collagenase, and elastase activity. Therefore, the composition of the present invention can be usefully used as a cosmetic composition for the purpose of antioxidant, whitening, wrinkle prevention and improvement related to the above activity.

In particular, since the excellent skin penetration effect of the composition of the present invention has been demonstrated by the nano-encapsulation technology using cubosome, it is expected to increase the action effect of the active ingredient by effectively penetrating it by applying it to the skin.

1 shows the analysis of the zeta potential value of monoolein for each manufacturer. (a; NU-CHEK-PREPmonoolein , b; NiKKOL MGO, c; Dermofeel ® PO, d; DUB OG)
Figure 2 shows the size distribution (a) and the zeta potential value distribution (b) in the dispersion of the nanoparticle liquid precursor containing PB17-lutein. The photograph included in graph (a) is a nanoemulsion of PBEN17.
Figure 3 shows the diffusion of PBEN17 through the ^{Strat-M ⓡ membrane.} The accumulated amount of lutein over time was represented by HPLC data.
4 shows the ORAC activity of PBEN17.
Figure 5 shows the activity inhibitory effect of xanthine oxidase of PBEN17.
6 shows the results of analyzing the DPPH radical antioxidant activity of PBEN17.
7 shows the results of analyzing the tyrosinase inhibitory activity of PBEN17.
8 shows the results of analyzing the collagenase inhibitory activity of PBEN17.
9 shows the results of analyzing the elastase inhibitory activity of PBEN17.
10 shows the skin irritation results for each formulation of PBEN17.

Hereinafter, the present invention will be described in more detail through preparation examples and experimental examples. These preparations and experimental examples are only for explaining the present invention in more detail, and it is common knowledge in the art that the scope of the present invention is not limited by these preparations and experimental examples according to the gist of the present invention. It will be self-evident for those who have it.

All data were expressed as mean±standard deviation and rounded off. A t-test was performed for the difference between the two means, and a post-test (Tukey's multiple range test) variance analysis was performed for the difference between three or more means, and a significant difference was tested at the p-value<0.05 level.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The detailed description of the present invention set forth below refers to the accompanying drawings, which show by way of illustration specific embodiments in which the present invention may be practiced. The scope of the present invention, if properly described, is limited only by the appended claims, along with all scope equivalents to those claimed.

Hereinafter, in order to enable those of ordinary skill in the art to easily practice the present invention, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Experimental Example 1. Microalgal Fermentation (PB17)

In the present invention, freshwater microalgae PB17 ( Auxenochlorella protothecoides, Chlorella minutissima ) was used. This strain was chosen because it contains large amounts of lutein. The strain was used for inoculation after storage in a cryogenic refrigerator at -80°C prepared as an optimal medium cell stock vial. The strains were cultured with shaking in 50L and 500L flasks, respectively, and used for inoculation after culturing for 24 hours. The medium used at this time was 4-5 g of K ₂ HPO ₄ , 3-4 g of NaH ₂ PO ₄ , 0.2-0.3 g of MgSO ₄ 7H ₂ O, 0.02-0.03 g of CaCl ₂ 2H ₂ O, 0.2-0.3 g citric acid, 2-3g MSG, 3-4g yeast extract, trace element (mineral) solution, vitamin solution. After setting the initial sugar concentration of the 5KL fermenter to 40 g / L, the initial rpm 40, air 1300 (0.4 vvm) temperature, 28 ℃, pH 6.8, PSP (Phycoil Signal Process; PCT / US2010/049347) state. It was cultured for about 140 hours (about 6 days) under optimal conditions (maximum rpm 60, air 1700 (0.57 vvm)).

Experimental Example 2. Pretreatment of microalgae (PB17) crude oil complex

Microalgae (PB17) were fermented using controlled lighting, and the crude complex was obtained using supercritical fluid extraction (SFE) technique. The extract was mixed 1:1 (v/v) with absolute ethanol as an extraction solvent. As a solvent, ethanol is generally recognized as a safe (GRAS) solvent according to the classification of the Ministry of Food and Drug Safety. These solutions were left in the dark at 55° C. for 18 hours with stirring. The solution was centrifuged at 2500 rpm for 15 minutes and the supernatant was collected. The collected supernatant was left at -20°C for 1 hour, then centrifuged at 2500 rpm for 15 minutes, and the supernatant was collected again. This step was repeated until almost no solution precipitated.

After performing these extractions and processing for HPLC analysis, this extraction solution was prepared for the formation of the nanoparticle encapsulation step.

Experimental Example 3. Preparation of PB17 encapsulated nanoparticles (PBEN17)

A liquid precursor was prepared by mixing monoolein, emulsifier and PB17 extract. For the preparation of the PB17 liquid precursor, Nikkol MGO (Nihonsurfactant kogyo k.k, Japan) monoolein was used. Monoolein preheated to 45° C. was completely dissolved in the PB17 ethanol extract. Monoolein was mixed with PB17 ethanol extract in a ratio of 1: 5 (v/v). As an emulsifier, polysorbate 80 was used in the formation of the liquid formulation. Polysorbate 80 was mixed with the preliminary mixture (mixture of monoolein and PB17 ethanol extract) at a ratio of 1:1.875. Most of the ingredients were added to the liquid mixture and stirred until clear. In some cases, the ethanol was evaporated for several hours to remove any traces of solvent and to control. These liquid precursors were dispersed by adding distilled water by vortexing the mixture for 2 minutes. These liquid precursors and water were mixed in a ratio of 1: 2.86 (v/v). The liquid precursor in water formed a dispersion of lipid particles.

Experimental Example 4. Particle Size Distribution Analysis and Zeta (ζ) Potential Measurement

Analysis of particle size was performed using dynamic light scattering technique. Average particle size and polydispersity index (PDI) were measured for all formulations using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) with a He/Ne laser (λ = 633 nm) and a scattering angle of 90°. Particle size measurement was performed on diluted lipid nanoparticles dispersed in water at 25°C. The measurement range of the Zetasizer Nono ZS90 is about 0.3 nm to 5 μm. Immediately, diluted samples were transferred to polystyrene cuvettes for sizing or to capillary cells for zeta (ζ) potential.

The zeta (ζ) potential is the potential between the particle surface and the dispersion in the sliding plane. The mobility u was transformed into a ζ potential using the Smouluchowski relationship: ζ = uη/ε. where η and ε are the viscosity and permittivity of the solution, respectively. The measurement of the PBEN17 sample was repeated 5 times.

Experimental Example 5. In vitro skin penetration study

In vitro permeation studies were performed with vertical Franz diffusion cells with an 8.0 ml capacity receptor compartment and 1.00 cm ^{2 diffusion area.} A synthetic (artificial) membrane (Strat-M ^ⓡ EMD Millipore, MA) was used.

The Strat-M ^ⓡ membrane was designed to mimic the layer structure and lipid chemistry of human skin. The thickness of each film is approximately 300 μm; a top layer supported by two layers of porous polyethersulfone (PES) on a single layer of polyolefin nonwoven support.

Each formulation was applied ^{to a Strat-M ⓡ} membrane with a glossy side in contact with the donor compartment. The acceptor fluid of each cell was filled with 80% ethyl alcohol or 100% ethyl alcohol to enable solubility, maintained at 37 °C ± 0.5 °C under synchronous continuous stirring using a magnetic stirrer at 600 rpm, O-rings were connected to the membrane and each The chamber holds the membrane. 1000 μl of each formulation was added to the donor compartment of each Franz diffusion cell and covered. Sampling of 800 μl was performed in each receptor chamber and replaced with fresh receptor fluid at 1, 2, 3, 4, 6, 20 and 24 hours. All samples collected were analyzed by HPLC regression equations obtained from standard curves prepared on the day of analysis. All formulations were tested in 10 replicates and data are reported as mean±SD.

The cumulative amount (Q) of the active compound released per surface of the membrane was obtained using the following equation.

는 샘플링 간격 1에서 n-1로 결정된 화합물(㎍/㎖)의 농도의 합이다. 샘플링 분취 량(0.8㎖) 및 A는 샘플 우물의 표면적이다. 이 연구에서 표면적은 1.00cm²였다.where Q is the cumulative amount of compound released per the surface of the membrane (μg/cm ² ), and Cn is the concentration of compound (μg/ml) determined at the nth sampling interval. V is the volume of an individual Franz diffusion cell and

is the sum of the concentrations of compounds (μg/ml) determined to be n-1 at sampling interval 1. Sampling aliquot (0.8 ml) and A are the surface area of the sample well. The surface area in this study was 1.00 cm ² .

Nanoparticle emulsions were prepared using a PBEN17 lutein mixture. PB17 was separated from water without mixing and permeabilized in Franz cell diffusion assays using ^{artificial Strat-M ⓡ membranes.}

As expected, a certain amount of lutein was observed in the receptor fluid. The PB17 encapsulated nanoparticle dispersion was filled with 80% ethyl alcohol in the receptor chamber. Miglyol® as a receptor solution for in vitro penetration studies 812/ethyl alcohol (1 : 9) (v/v) was used.

In the penetration of PBEN17, the cumulative amount increased with time, and the cumulative amount of lutein diffusion was about 2.5 μg/cm ² , with a penetration rate of approximately 30% at 24 hours.

PB17 encapsulated nanoparticles (PBEN17) Cumulative amount(㎍/cm ² ) Penetration rate (%) 1H 0.02 ± 0.00 0.30 ± 0.00 2H 0.08 ± 0.00 1.17 ± 0.06 3H 0.17 ± 0.06 2.38 ± 0.84 4H 0.33 ± 0.00 4.57 ± 0.04 6H 0.60 ± 0.00 8.38 ± 0.06 20H 2.25 ± 0.01 31.36 ± 0.16 24H 2.35 ± 0.10 32.67 ± 1.32

Experimental Example 6. High Performance Liquid Chromatography (HPLC) Analysis

Lutein was quantified using validated HPLC methods. The HPLC analysis conditions for analyzing the samples obtained through the Franz diffusion cell are as follows. For the separation of lutein, a chromatography column (SunFire ^ⓡ C18; 5 μm, 4.6 X 250 mm, Waters Corporation, Milford, MA, USA) was used. For the YL9100 system, HPLC, YL9101 vacuum degasser, YL9110, YL9131 column compartment, YL9160 PDA detector, and YL9150 autosampler (Yonginchromass, Korea) were used for the vacuum degasser. Methanol was used as the mobile phase, and the flow rate was 1 ml/min. The detection wavelengths were 446 nm and 474 nm.

Lutein (α-Carotene-3,3′-diol, β, ε-carotene-3,3′-diol, β,ε-caroten), ethyl alcohol, methanol and HPLC grades were purchased from Sigma-Aldrich.

Experimental Example 7. Analysis of Oxygen Radical Absorption Capacity (ORAC)

The ORAC assay is a widely used method to measure the antioxidant capacity of various samples. The principle of ORAC analysis is that the total antioxidant activity of a sample is measured through a complete oxidation reaction. The final concentrations of the samples (0.01, 0.1, 1, 5, 10%) were diluted with sodium phosphate buffer (75 mM, pH 7.4) and 20 μl of this sample was added to a black well plate and 20 μl Trolox (Cat No. 648471, Sigma). ; 0.1, 0.4, 1.2, 3.7, 11.1, 33.3, 100 μM) were added to the black well plate. 120 μl of fluorescein solution (117 nM) was received in each well and incubated at 37° C. for 15 minutes. 60 μl of AAPH (2,2'-Azobis-2- amidinopropane, Cat No. 100110, Sigma) solution (40 mM) was added to each well. The decrease in fluorescence was measured every minute for 90 minutes using a fluorometer (excitation 485 nm / emission 520 nm). ORAC values were expressed as 'μM Trolox equivalent' using a regression curve between the standard reagent Trolox concentration and AUC.

Oxygen radical absorption capacity (ORAC) measurements showed that among the individual positive controls such as 0.01% ascorbic acid, arbutin, ascorbylglucoside, lutein and astaxanthin, astaxanthin had the highest ORAC value and 17.3 uM Trolox equivalent (TE). This means that the peroxyradical scavenging ability is stronger (p <0.01). Ascorbylflucoside, ascorbic acid, arbutin and lutein were measured as 13.3, 13.0, 13.0, 12.9 uM TE in the order, all positive controls exhibited significant peroxyl radical scavenging activity compared to the vehicle group (p < 0.01). PBEN17 was measured as 5.0, 6.5, 12.7, 13.3, and 13.4uM TE at concentrations of 0.01, 0.1, 1, 5, and 10%, respectively, and all groups except 0.01% showed significant peroxyradical scavenging activity compared to the vehicle group. shown (p <0.05). In addition, more than 5% of PBEN17 showed higher peroxyradical scavenging activity than the positive control. For PBEN17, the ORAC value also tended to increase with increasing concentration (p <0.05).

Experimental Example 8. Xanthine oxidase inhibition assay

Long-term exposure to UV radiation leads to the formation of reactive oxygen species (ROS), which leads to abnormal cell function and consequently skin aging. Melanogenesis produces hydrogen peroxide and ROS in melanocytes. ROS are also produced in xanthine oxidase catalysis. An experiment was performed to confirm the inhibition of ROS production through the inhibition of xanthine oxidase. The final concentration of the sample (0.01, 0.1, 1, 5, 10%) was diluted with sodium phosphate buffer (75 mM, pH 7.4) and 127 μl of this sample was added to a UV microplate. 7 μl of xanthine oxidase (Cat No. 10110434001, Sigma, 0.1 U/ml) was placed in each well and incubated at 37° C. for 10 minutes. 66 μl of a xanthine (Cat No. X0625-5G, Sigma, 400 μM) mixture was incubated in each well at 37° C. for 10 minutes and measured at 295 nm.

As a result of measuring xanthine oxidase inhibitory activity, 0.01% ascorbic acid, arbutin, ascorbyl glucoside, lutein and astaxanthin used as positive controls did not show significant inhibitory activity of xanthine oxidase. In the case of PBEN17, the 5% treatment group showed a significant inhibitory activity of 15% (p <0.05), and the inhibitory activity increased with increasing concentration.

Experimental Example 9. DPPH analysis (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method

The DPPH assay can be applied as a standard method for evaluating antioxidant capacity. The antioxidant activity can be assessed by the reduction of the purple color of the stable free radical DPPH. A final concentration of 80 μl of sample (0.01, 0.1, 1, 5, 10%) was diluted with absolute ethanol and 80 μl of 0.2 mM DPPH (2,2-diphenyl-1-picryl-hyerazyl-hydrate, Cat. No. 14805). , Cayman, USA) was diluted in absolute ethanol and mixed. When the reaction stabilized after 30 min at RT, the absorbance decrease at 520 nm was measured using a microplate reader.

As a result of measuring the DPPH radical scavenging activity, ascorbic acid had the highest DPPH radical scavenging activity among individual positive controls such as 0.01% ascorbic acid, arbutin, ascorbyl glucoside, lutein and astaxanthin, and 89.4% compared to the vehicle group. It showed strong radical scavenging ability. The radical scavenging activity of ascorbyl glucoside, arbutin and astaxanthin was observed in the order of 78.8, 31.0, and 23.0%, respectively, which was significantly increased compared to the vehicle group (p <0.05). For PBEN17, the 5 and 10% treatment groups showed significant radical scavenging activity of 9.1 and 9.3%, respectively, compared to the vehicle group (P <0.05).

Experimental Example 10. Tyrosinase inhibition assay

Tyrosinase is known as the enzyme responsible for the production of melanin in mammals. Tyrosinase inhibition experiments may predict the effect of whitening. A final concentration of 180 μl of a sample (0.01, 0.1, 1, 5, 10%) was diluted with 100 mM phosphate buffer (pH 6.8) and placed in the wells of a microplate. 10 μl of 2000 U/ml mushroom tyrosinase (Cat No. T3824, Sigma) was added to each well and incubated at 37° C. for 6 minutes. Then, 10 μl of DOPA (L-Dopa-phenyl-d3, Cat. No. 333786, Sigma, 10 mM) was added to each well and incubated at 37° C. for 1 minute. Then, the absorbance of dopachrome was measured at 475 nm using a microplate reader.

Tyrosinase inhibitory activity was measured among 0.01% ascorbic acid, arbutin, ascorbylglucoside, lutein and astaxanthin used as positive controls, and ascorbic acid showed the highest tyrosinase inhibitory activity at 43.1% compared to the vehicle group. , followed by lutein (39.8%) showed significant tyrosinase inhibitory activity (p <0.05). In the case of PBEN17, the 5 and 10% treatment groups showed significant tyrosinase inhibitory effects of 20.5 and 33.9%, respectively, compared to the vehicle group (p <0.05).

Experimental Example 11. Collagenase inhibitor analysis

Collagenase production is one example and inhibition of wrinkle improvement by collagenase may predict the efficacy of wrinkle improvement. 4 mM CaCl2 was added to 0.1M Tris-HCl buffer (pH 7.5). The substrate solution in which 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-Arg (Cat No. 89064, Sigma, 0.3 mg/ml) was dissolved was dissolved and collagenase (0.2 mg/ml) was added to the sample mixture. did. The vials were incubated at room temperature for 20 minutes. The reaction was stopped by adding 6% citric acid (Cat No. 791725, Sigma) to each vial. Ethyl acetate was added to each vial, and each supernatant was placed in a UV microplate and measured at 320 nm.

As a result of measuring the collagenase inhibitory activity, 0.01% ascoric acid, arbutin, ascorbyl glucoside, lutein and astaxanthin used as positive controls did not show any significant difference. In the case of PBEN17, the 1, 5, and 10% treatment groups showed significant collagenase inhibitory activity of 65.9, 89.8, and 94.3% (p <0.01), respectively, compared to the vehicle group, and the collagenase inhibitory activity was increased with increasing.

Experimental Example 12. Elastase Inhibition Analysis

Elastase is an enzyme that induces wrinkle formation by decomposing elastin in the stratum corneum of the skin and reducing skin elasticity. Therefore, the elastase inhibition experiment is meaningful in confirming the wrinkle improvement ability. A final concentration of 100 μl of sample (0.01, 0.1, 1, 5, 10%) was diluted with 50 mM Tris-HCl buffer (pH 6.8) and placed in the wells of a microplate. 50 μl of elastase (Cat No. E0127, Sigma, 0.2 U/ml), 10 μl of N-succinyl-(Ala)3-p-nitroanilide (Cat No. S476, Sigma, 1 mg/ml) in each well added and incubated at 37°C for 30 min. Measurements were made at 410 nm using a microplate reader.

As a result of measuring the elastase inhibitory activity among 0.01% ascorbic acid, arbutin, ascorbylglucoside, lutein and astaxanthin used as a positive control, lutein had the highest elastase inhibition by 53.6% compared to the vehicle group. showed activity. The elastase inhibitory activities of astaxanthin, arbutin, and ascorbic acid were observed in the order of 53.6, 30.9, 6.25, and 5.7%, respectively, which significantly increased compared to the vehicle group (p <0.05). For PBEN17, the 0.01, 0.1, 1, 5, and 10% treatment groups showed significant elastase inhibitory activity of 6.2, 7.3, 23.3, 57.3, and 65.3%, respectively, compared to the vehicle group (p <0.01). The elastase inhibitory activity increased with increasing concentration.

Experimental Example 13. Skin irritation test

After the tissue was incubated for 42 h, the entire medium was removed using a micropipette. 200 μl of MTT solution (0.4 mg/ml), Keraskin™-VM (Biosolution Co., Ltd., Korea) was transferred to a 24-well plate in a 24-well plate, and 100 μl of MTT solution was received inside the insert. Incubated at 37° C., 5% CO _{2 in an} incubator for 3 hours ± 5 minutes. Light was blocked. To extract the formazan, Keraskin™-VM was transferred to a new 6-well plate pre-filled with 1.9 ml isopropanol. 0.1 ml of isopropanol was received inside the insert and formazan was extracted for 3 hours ± 5 minutes.

Through this, formazan was bound to the inside and outside of the insert using a micropipette, pipetting in a 6-well plate. After extraction of 250 μl of formazan, each tissue was transferred to a 96-well plate, and each well was checked for air bubbles. Optical density (OD) was measured at 570 nm using a spectrophotometer.

Cell viability was calculated based on the absorbance values of negative viable DPBS. The solvent control (OD _blank ) was calculated as the average value of _{the OD blank} of each plate tissue. Negative control (OD _NC ) was calculated as the average of negative control absorbance values (OD _NCraw )-OD _{blank for each plate (ODblank).}

OD _NC (%) = OD _NCraw - OD _blank

The cell viability of the negative control is 100%. The positive control (OD _PC ) was calculated as the average of the OD _blank from the positive control absorbance values (OD _PCraw ) for each plate (OD _PC).

OD _PC = OD _PCraw - OD _blank

A positive control was obtained by dividing _{the mean OD PC} values of the four treated tissues by the mean OD _{NC values.}

Cell viability (%) of positive control = (mean OD _PC / mean OD _NC ) X 100

The test material (OD _T ) was averaged by subtracting the OD _blank from each test material absorbance value (OD _Traw ) for each plate (OD _{T ).}

OD _T = OD _Traw - OD _blank

In addition, functional check values were subtracted for colored and reducing substances.

OD _T = OD _Traw - OD _blank -OD _C or OD _R

Cell viability was obtained by dividing the mean ODT value of the tissues treated with the test substance by the mean OD _{NC value.}

Cell viability (%) of test substance = (mean OD _R / mean OD _NC ) X 100

Non-stimulated (> 50%)/stimulated (≤ 50%) determined based on survival cutoff 50%

As a result of skin irritation test using the Korean skin model KeraSkinTM-VM, 9 samples were evaluated as having no skin irritation with a cell viability of 50% or more in the artificial skin model after application of the test substance. In this study, five prototype PB17 lotion formulation (PB17-LO), PB17 cream formulation (PB17-CR), skin irritation evaluation formulation of PB17 encapsulated nanoparticle lotion (PBEN17-LO), PB17 encapsulated nanoparticle cream formulation (PBEN17-CR) and PB17 spray formulation (PB17-SP) artificial skin models were used. As a result of all samples, 50% or more of non-stimulatory, cell viability was confirmed. Therefore, all samples are free from skin irritation.

Experimental Example 14. Identification of Lutein Fermentation Microalgae (PB17)

Microalgal PB17 was cultured in optimal medium using controlled lighting (PSP; Phycoil Signal Process; PCT/US2010/049347), and crude fat was obtained using supercritical fluid extraction (SFE) technique.

Lutein in the crude fat mixture was analyzed by Phycoil Biotech Korea. As a result of HPLC analysis of the content of lutein from fermented PB17 subjected to supercritical fluid extraction, the content was about 200 ppm. A supercritical fluid extraction (SFE) crude fat mixture was pretreated by mixing with absolute ethanol as an extraction solvent, and the content of the lutein unprecipitated solution was analyzed by HPLC. The content percentage of lutein contained in the pretreated PB17 crude fat mixture was about 45%, and the concentration of lutein was 350 ppm.

Experimental Example 15. Analysis of particle size and zeta (ζ) potential of PBEN17

The liquid precursor in water formed a dispersion of PB17 liquid nanoparticles ranging in diameter from 100 to 200 nm. In a preliminary study, several types of monoolein for forming nanoparticles and stability were tested because monoolein (NU-CHEK-PREP, INC, USA, Cat No. 111-03-5) is expensive for commercial use. Monoolein types for nanoparticle formation were NiKKOL MGO (Nihon surfactant kogyo, Japan, Cat No. 25496-72-4), Dermofeel ^® PO (drstraetmans, Germany, Cat No. 25496-72-4, 111-03-) 5 or 91744-09-1), and DUB OG (Glycerol oleate, France, Cat No. 68424-61-3). They are relatively inexpensive compared to monoolein (NU-CHEK-PREP, INC.). Three types of monooleins were compared to NU-CHEK-PREP monooleins in forming nanoparticles without encapsulating molecules. Using NiKKOL MGO, Dermofeel ^® PO, and DUB OG, dispersed liquid precursors can be obtained as when using NU-CHEK-PREP monoolein, and the three types have similarly excellent zeta potential values. It was confirmed that NiKKOL MGO, Dermofeel ^® PO and DUB OG can replace NU-CHEK-PREP monoolein.

For liquid nanoparticle formation containing PB17, no monoolein Dermofeel ^® PO was used to form the nanoparticle component. First, in the case of A, the nanoparticle size was unstable as the precursor was generated or its content was adjusted with time. Nanoparticles containing PB17 could not be optimized with ^{Dermofeel ® PO.}

It was decided to use an encapsulated nanoparticle emulsion containing PB17 using Nikkol MGO instead of monoolein (NU-NUCK-PREP, INC). PBEN17 was prepared as a liquid precursor with an optimized content ratio of Nikkol MGO: PB17 extract: polysorbate 80 as 14.7 (v/v): 73.5 (v/v): 11.8 (v/v). A particle size (diameter) of 100-200 nm and a zeta potential value (-33.2 mV) were obtained and were stable over time.

Korea Institute of Bioscience and Biotechnology KCTC18635P 20171124

Claims (12)

Nano-sized cubosome crystals containing lutein extracted by supercritical method by fermenting and culturing microalgae specified by accession number KCTC 18635P by PSP method.
delete The method of claim 1 .
The cubosome is a crystal, characterized in that composed of monoolein. The method of claim 1 .
The cubosome is a crystal, characterized in that it contains lutein (lutein, PB17 culture extract) at a concentration of 100 ~ 500 μg / ml, and monoolein dispersion at a concentration of 0.1 ~ 0.5 mg / ml. Fermenting and culturing microalgae of accession number KCTC 18635P by the PSP method;
extracting lutein by a supercritical method;
preparing a lutein dispersion;
preparing a monoolein dispersion;
mixing the lutein dispersion and the monoolein dispersion;
filtering the mixed solution through a fine separation network;
A method for producing cubosome nanoparticle crystals comprising the step of centrifuging when a precipitate is formed in the mixed solution.
delete 6. The method of claim 5,
The method for producing a cubosome nanoparticle crystal, characterized in that the fine separation network is a filter having a size of 0.1 to 0.5 micrometers. A cosmetic composition comprising, as an active ingredient, nano-sized cubosome crystals containing lutein extracted by supercritical method by fermenting and culturing microalgae specified by accession number KCTC 18635P by PSP method.
delete 9. The method of claim 8.
The cubosome is a cosmetic composition, characterized in that consisting of monoolein. 9. The method of claim 8,
The cubosome is a cosmetic composition comprising lutein (PB17 culture extract) at a concentration of 100 to 500 μg/ml, and monoolein dispersion at a concentration of 0.1 to 0.5 mg/ml. 9. The method of claim 8,
The cosmetic composition is a cosmetic composition, characterized in that for antioxidant, whitening, or wrinkle improvement.

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