KR102266329B1 - Nano-liposome Comprising Idebenone and Bisabolol - Google Patents

Abstract

The nano liposome of the present invention has a complex including idebenone and bisabolol. Here, since bisabolol dissolves poorly soluble idebenone, it not only enhances the skin penetration effect of idebenone, but also exhibits an antioxidant action on its own, thereby synergistically with idebenone to exhibit a whitening or moisturizing pharmacological action. Therefore, the nano-liposome of the present invention has excellent transdermal delivery rate of idebenone and can be used as a cosmetic formulation with excellent whitening or moisturizing effect.

Description

Nano-liposome containing idebenone and bisabolol {Nano-liposome Comprising Idebenone and Bisabolol}

The present invention relates to a nano-liposome containing idebenone, and more particularly, to a cosmetic composition for whitening or moisturizing comprising the nano-liposome as an active ingredient.

Idebenone is a ubiquinone antioxidant that exists in living cell membranes and has a structure similar to coenzyme Q10, which is mainly involved in the Electron Transfer Chain (ETC) involved in the generation of ATP (Adenosine triphosphate), the cellular energy in the mitochondria. The chemical name is 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone [dimethoxy-5-methyl-6-(10-hydroxy decyl)- 1,4-benzoquinone] and the molecular weight is 338.44, and it is known as a purified yellow crystalline powder material mainly obtained by chemical synthesis.

According to known research papers, it has been reported in several academic papers that medicines and cosmetics applied with idebenone for pharmaceutical or cosmetic use have very good anti-aging effects.

In addition, idebenone is effective in cell activation by participating in ETC (Electron Transfer Chain) related to the generation of ATP (Adenosinetriphosphate), which is cellular energy in the mitochondria, and in mitochondria, reactive oxygen species (ROS) It protects the skin by preventing formation and medically promotes Nerve Growth Factor (NGF), which is a degenerative neurological disease that is a problem due to aging, Alzheimer's disease, and cerebrovascular disease, a vascular disease. disease) and liver disease, the results of studies have also been reported.

However, idebenone, one of these bioavailable active substances, has a complex structure such as a quinone group, a saturated hydrocarbon group, and a hydroxyl group, and has a high polarity and a low melting point. Therefore, in the emulsion system, stabilization is difficult and skin penetration is not easy, so it is difficult to apply it to actual cosmetics.

A method of transdermal formulation of idebenone by encapsulating liposome has been attempted (Registration Patent No. 10-2004147), but it is difficult to increase transdermal absorption in the form of simply encapsulating in the inner aqueous phase of the liposome, and it is difficult to increase the stability of the liposome. It was difficult to formulate in a form.

In this situation, the present inventors have discovered that the two active ingredients, idebenone and bisabolol, can achieve effects such as whitening and moisturizing mutually synergistically, so that the first active ingredient, idebenone, is used as the second active ingredient, bisabolol. As it is dissolved in a solvent containing a phospholipid and inserted into the phospholipid bilayer, the active ingredient is inserted into the liposome in a relatively small amount to maintain the stability of the liposome, and a formulation capable of achieving an excellent effect was devised.

Technical problem to be achieved by the present invention is a phospholipid bilayer; And it is to provide a nano-liposome comprising a complex comprising a idebenone (idebenone) and bisabolol (bisabolol).

Another technical problem to be achieved by the present invention is to provide a cosmetic composition for whitening or moisturizing comprising the nano-liposome as an active ingredient.

Another technical problem to be achieved by the present invention is to provide a method for preparing the nano-liposome.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

In order to achieve the above technical object, an embodiment of the present invention is a phospholipid bilayer; And idebenone (idebenone) and bisabolol (bisabolol), including a complex comprising a, provides a nano-liposome.

Here, since bisabolol dissolves poorly soluble idebenone, it not only enhances the skin penetration effect of idebenone, but also exhibits an antioxidant action on its own, thereby synergistically with idebenone to exhibit a whitening or moisturizing pharmacological action. Therefore, the nano-liposome of the present invention has excellent transdermal delivery rate of idebenone and can be used as a cosmetic formulation with excellent whitening or moisturizing effect.

The term “nanoliposome” used in the present invention is a nano-sized spherical particle composed of a phospholipid bilayer, in particular, as one of the most appropriate colloidal particle formulations applicable to the human body due to excellent biocompatibility of the phospholipids constituting the liposome membrane. is considered Since poorly water-soluble drugs often have affinity for the hydrophobic portion, they can be solubilized by inserting them between the phospholipid molecules constituting the double membrane, so that liposome-based products have been researched and developed as poorly soluble drug solubilizing formulations. However, when the drug is inserted into the liposome phospholipid membrane, it is difficult to secure sufficient space between the phospholipid molecules, so the insertion concentration of the hydrophobic drug is not high. In particular, aggregation and fusion of liposomes are the main causes of destabilization of liposomes. That is, when the hydrophobic drug is inserted into the membrane at a high concentration, aggregation or fusion of the liposome is deepened, so that the drug may be precipitated out of the lipid double membrane during storage after preparation or before reaching the site of action after administration. In this regard, the present inventors have discovered that the two active ingredients, idebenone and bisabolol, can synergistically achieve the effects of whitening and moisturizing, etc., so that the first active ingredient, idebenone, is used as the second active ingredient, bisabo. It was dissolved or dispersed in a solvent including a roll and inserted into the phospholipid bilayer. Accordingly, in the nano-liposome of the present invention, a relatively small amount of active ingredient is inserted into the liposome, and excellent cosmetic effects can be achieved while maintaining the stability of the liposome.

As used herein, the term “idebenone” is an ubiquinone-based antioxidant present in living cell membranes and an electron transfer system (ETC, Electron Transfer) related to the generation of ATP (Adenosinetriphosphate), which is cellular energy in the mitochondria. Chain) as a chemical substance having a structure similar to coenzyme Q10 mainly involved in 4-benzoquinone [dimethoxy-5-methyl-6-(10-hydroxy decyl)-1,4-benzoquinone], the molecular weight is 338.439 g mol ^-1 , and purified yellow crystals obtained mainly by chemical synthesis It is known as a sex powder substance.

[Formula 1]

Idebenone is known to improve oxidation in mitochondria, an organ involved in cellular respiration, and to play a protective role in cellular oxidative stress. In the past, idebenone was mainly used for medicinal purposes because it has effects such as recovery of neurological symptoms such as ataxia and dementia due to nerve cell damage, improvement of hearing loss caused by external damage, and improvement of optic neuritis. As research papers have been published that it is effective, it is also used for anti-aging, skin moisturizing, and improvement of fine wrinkles by promoting collagen synthesis.

As used herein, the term “Bisabolol” is one of natural monocyclic sesquiterpene alcohols, and is also referred to as alpha-bisabolol (α-(-)-bisabolol) or levomenol. is known Alpha-bisabolol (α-(-)-bisabolol) has the structure of Formula 2 below, and the chemical name is 6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-2 -ol, and the molecular weight is 222.372g·mol ^-1 . Bisabolol is also known as a colorless, viscous oil that is the main component of the essential oil of the Brazilian Candeia tree ( Eremanthus erythropappus or Vanillosmopsis erythoropappa ) or German chamomile ( Matricaria recutita). Most commercially available bisabolols are prepared by distillation of Candeia bark. Bisabolol can also be synthesized chemically, can be obtained using various extraction methods known in the art, and if necessary, can be obtained through an additional purification process.

[Formula 2]

According to one embodiment of the present invention, the nano liposome of the present invention contains idebenone, which is effective in whitening, anti-aging and wrinkle improvement, as an active ingredient, and bisabolol, an active ingredient known to have antioxidant effects, of idebenone. used as a solvent. The present inventors confirmed that when idebenone is dissolved in a bisabolol solvent and treated with (melanoma) cells, it synergistically reduces melanin production capacity and suppresses related gene expression compared to the case of treating each separately. (Experimental Example 1).

As used herein, the term “phospholipid bilayer” refers to a membrane composed of two layers of phospholipids having a hydrophilic head and a hydrophobic tail. Typically the hydrophilic heads are exposed on the surface and inside of the bilayer, and the hydrophobic tail of the phospholipid bilayer is not exposed. The phospholipid bilayer is composed of a hydrophilic head region exposed on the liposome surface, a hydrophobic tail region, and a hydrophilic head region exposed inside the liposome. The complex comprising idebenone and bisabolol according to the present invention may be inserted into the phospholipid bilayer. That is, the complex may be inserted between both hydrophilic head regions.

According to one embodiment of the present invention, in the phospholipid bilayer, the phospholipid constituting the phospholipid bilayer may be at least one selected from the group consisting of neutral lipids, anionic phospholipids and cationic phospholipids. The neutral lipid is La-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine 1,2-distearoyl-sn-glycero-3-phosphocholine 1,2-di It may be at least one selected from the group consisting of palmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The anionic phospholipid is L-α-phosphatidic acid, L-α-phosphatidyl-DL-glycerol, cardiolipin, La-phosphatidylinositol, 1,2-dilauroyl-sn-glycero-3-[phospho -rac-(1-glycerol)], 1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], 1,2-dilauroyl-sn-glycero-3 -Phosphate, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac- (1-glycerol)], 1,2-dimyristoyl-sn-glycero-3-[phospho Pho-L-serine], 1,2-dimyristoyl-sn-glycero-3-phosphate, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol) )], 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dipalmi Toyl-sn-glycero-3-[phospho-L-serine], 1,2-dipalmitoyl-sn-glycero-3-phosphate, 1,2-disteroyl-sn-glycero-3- [Phospho-rac- (1-glycerol)], 1,2-disteroyl-sn-glycero-3-[phospho-L-serine], 1,2-disteroyl-sn-glycero- 3-phosphate, 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy (polyethylene glycol 2000), 1,2-disteroyl-sn-glycero-3-phospho Phoethanolamine-N-[maleimide (polyethylene glycol) 2000], 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[PDP (polyethylene glycol) 2000], 1-palmi Toyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)], 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-L- Serine], 1-palmitoyl-2-oleyl-sn-glycero-3-phosphate and oleic acid may be at least one selected from the group consisting of. The cationic phospholipid is 1,2-dioleoyl-sn-glycerol. Rho-3-ethylphosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, dioleoyl glutamide, distearoyl glutamide, dipalmitoyl glutamide, dioleoyl amine Spartamide, 1,2-Dioleoyl-3-dimethylammonium-propane, 3-β-[N-(N′,N′-dimethylaminoethane)carbamoyl ]Cholesterol, methyldioctadecylammonium bromide, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine And it may be at least one selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.

According to one embodiment of the present application, the nano-liposome of the present invention may include 1 to 10 parts by weight, specifically, 1 to 5 parts by weight, based on 1 part by weight of idebenone, the phospholipid.

As used herein, the term “complex” refers to a solution in which idebenone as a solute is dissolved or dispersed in a solvent containing bisabolol. In the complex, idebenone is uniformly mixed with the solvent containing bisabolol, and the particles of idebenone are surrounded by the solvent particles containing bisabolol and have a stable state. In this regard, the present inventors have confirmed that idebenone has a high solubility of about 34% at room temperature with respect to bisabolol. A solution in which idebenone is dissolved in bisabolol below the above solubility means that idebenone does not precipitate as particles having a size of 1 μm or more, specifically 100 nm or more, and more specifically 50 nm or more. The complex may include an additional solvent to increase the dissolution or dispersibility of idebenone. Specifically, solvents such as alcohols and/or polyhydric alcohols may be used. The alcohol may be at least one selected from the group consisting of methanol, ethanol, isopropanol, and butanol, and the polyhydric alcohol may be at least one selected from the group consisting of glycerol, propanediol, and 1,2-hexanediol.

According to a preferred embodiment, the complex may be a mixture of 1 part by weight of idebenone and 1 to 10 parts by weight of bisabolol. Specifically, the complex may be a mixture of a solvent containing 1 part by weight of idebenone and 2 to 4 parts by weight of bisabolol. More specifically, a solvent containing 1 part by weight of idebenone and 2.5 to 3.5 parts by weight of bisabolol may be mixed. The present inventors confirmed that when mixed in the above ratio, a uniform complex was formed, while exhibiting excellent efficacy related to whitening and moisturizing to cells.

According to one embodiment of the present invention, the nanoliposome of the present invention may be one in which a complex in which idebenone is dissolved in a solvent containing bisabolol is inserted between phospholipid molecules in a phospholipid bilayer. The present inventors have applied a formulation in which idebenone is dissolved in a bisabolol solvent in a phospholipid bilayer of liposomes to artificial skin (Merck's Strat-M) using Franz cells. As a result, the transdermal absorption rate of idebenone, especially the dermis It was confirmed that the transmittance could be increased (Experimental Example 2).

According to one embodiment of the present application, the phospholipid bilayer may include an anionic surfactant and a fatty acid.

As used herein, the term “anionic surfactant” refers to a surfactant in which the hydrophilic moiety has a negative charge. According to one embodiment of the present application, the anionic surfactant is included in the phospholipid bilayer of the nano-liposome, so that the negatively charged site of the anionic surfactant may be present on the surface of the nano-liposome. In this case, the transdermal permeation efficiency may be improved. The present inventors confirmed that the transdermal absorption efficiency of nanoliposomes can be increased by additionally using an anionic surfactant (Experimental Example 2-1).

Specifically, the anionic surfactant is potassium cetyl phosphate (Potassium Cetyl Phosphate), sodium lauryl sulfate (Sodium Lauryl sulfate) sodium dodecyl sulfate (Sodium dodecyl sulfate), sodium laureth sulfate (Sodium Laureth sulfate), and sodium di It may be at least one selected from the group consisting of oxycholate (sodium deoxycholate). More specifically, the anionic surfactant may be potassium cetyl phosphate (Potassium Cetyl Phosphate), which is preferably used because it does not show irritation to the skin than other anionic surfactants.

According to one embodiment of the present application, the nano-liposome of the present invention may include 1 to 10 parts by weight, specifically, 1 to 5 parts by weight of the anionic surfactant based on 1 part by weight of idebenone.

As used herein, the term “fatty acid” means that the terminus of an aliphatic hydrocarbon is a carboxyl group. According to one embodiment of the present application, the fatty acid is included in the phospholipid bilayer of the nano-liposome, so that the carboxyl group may be present on the surface of the nano-liposome. In this case, it can serve to not only give elasticity to the liposome membrane, but also to prevent oxidation. The present inventors confirmed that the transdermal absorption efficiency of nanoliposomes, particularly the dermal permeability, could be significantly increased by additionally using a fatty acid (Experimental Examples 2-2 and 3). According to one embodiment of the present application, the fatty acid is elaidic acid, lauric acid, limistic acid, palmitic acid, stearic acid, linoleic acid, oleic acid, linolenic acid, arachidonic acid, and caprylic/capric triglyceride ( MCT) may be one or more selected from the group consisting of.

According to one embodiment of the present application, the nano-liposome of the present invention may include 0.1 to 1.0 parts by weight, specifically 0.5 to 1.0 parts by weight, of the fatty acid based on 1 part by weight of idebenone.

According to one embodiment of the present invention, an aqueous phase may exist in the inner space of the nano-liposome of the present invention. The aqueous phase may contain a water-soluble active ingredient such as a polar molecule in a dissolved form, and may be encapsulated in a state in which the fat-soluble active ingredient is not dissolved.

As one aspect for achieving the technical problem of the present invention, the present invention provides a cosmetic composition for whitening or moisturizing comprising nano liposomes as an active ingredient.

The description of the nano-liposome is the same as described above.

The nano-liposome of the present invention can exhibit a whitening effect when applied to the skin, which is due to the fact that idebenone of the nano-liposome exhibits high antioxidant power, and bisabolol inhibits the expression of whitening-related enzymes. Specifically, idebenone prevents oxidation by improving oxidative metabolism at the mitochondrial level and protecting cells against oxidative stress. Specifically, idebenone is analogous to CoQ10, but the antioxidant effect is 6 times stronger than that of CoQ10. Accordingly, idebenone can be applied to skin cells to suppress the production of melanin and prevent skin aging. In addition, bisabolol inhibits the expression of enzymes such as tyrosinase, TRP-1 and TRP-2.

The nano liposome of the present invention can be applied to the skin to exhibit moisturizing and anti-wrinkle effects. This is because idebenone is effective in moisturizing the skin, and bisabolol helps relieve skin irritation. Specifically, idebenone has been shown to have a moisturizing effect and improvement of fine wrinkles by promoting collagen synthesis, and bisabolol is known to help relieve and calm skin irritation.

The cosmetic composition of the present invention may additionally include conventionally added ingredients such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and conventional adjuvants and carriers to achieve a whitening or moisturizing effect.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil , powder foundation, emulsion foundation, wax foundation and spray, etc., but are not limited thereto.

More specifically, lotions such as flexible lotions or nourishing lotions, emulsions such as facial lotions and body lotions, creams such as nourishing creams, moisture creams, and eye creams, essences, cosmetic ointments, sprays, gels, packs, sunscreens, makeup It may be prepared in formulations such as foundations such as base, liquid type, solid type or spray type, powder, cleansing cream, cleansing lotion, makeup remover such as cleansing oil, cleansing agent such as cleansing foam, soap, body wash, and the like. In this case, the cosmetic composition is a surfactant, an emulsifier, a soap acid, a solvent, a colorant, a preservative, an antioxidant, an antifoaming agent, an antibacterial agent, an anti-redeposition agent, an enzyme, a plant or mineral oil, a fat, a fluorescent substance, a fungicide, a hydrophobic inducing substance, It may contain excipients including moisturizing agents, fragrances, fragrance carriers, preservatives, proteins, silicones, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, waxes, and the like.

When the formulation of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as a carrier component can be used

When the formulation of the cosmetic composition is a powder or a spray, toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propellants such as propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used.

When the formulation of the cosmetic composition is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid as carrier components Amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters can be used.

As another aspect for achieving the technical problem of the present invention, the present invention,

providing an idebenone dissolving portion comprising idebenone and bisabolol; providing a lipid lysate comprising phospholipids; And it provides a method for preparing a nano-liposome suspension comprising the step of mixing the idebenone dissolving unit and the lipid dissolving unit.

The terms used herein are the same as described above.

The properties of the nano-liposomes contained in the suspension prepared according to the preparation method are as described above.

The idebenone dissolving part containing idebenone and bisabolol is provided by dissolving idebenone in a solvent containing bisabolol. Specifically, bisabolol may be mixed with idebenone at room temperature and then stirred. According to one embodiment of the present application, the idebenone dissolving unit may further mix alcohol and / or polyhydric alcohol. The alcohol may be at least one selected from the group consisting of methanol, ethanol, isopropanol, and butanol, and the polyhydric alcohol may be at least one selected from the group consisting of glycerol, propanediol, and 1,2-hexanediol.

The lipid dissolving portion is provided by dissolving phospholipids in a solvent. Specifically, after dispersing the phospholipids in a solvent, it may be provided by stirring at a high temperature. The lipid dissolving unit may be to stir the phospholipid mixture at 60 to 90 ℃. In this case, the above-mentioned fatty acid and anionic surfactant may be additionally added. It may include a step of cooling after stirring. The solvent of the lipid dissolving part may be one or more solvents selected from the group consisting of glycerin, propylene glycol, and butylene glycol. In addition, preservatives such as phenoxyethanol may be additionally included in the lipid dissolving part of the present application.

After the two steps, the idebenone dissolving unit and the lipid dissolving unit are mixed and stirred to prepare liposomes.

According to one embodiment of the present invention, the preparation method of the present invention may further comprise adding water to the liposome to provide a liposome suspension.

The composition ratio (weight ratio of phospholipids, anionic surfactants, fatty acids, etc. based on parts by weight of idebenone) of the final product, nanoliposome, can be easily controlled by adjusting the amount added to each dissolution part in the manufacturing step. .

The present inventors discovered that two active ingredients, idebenone and bisabolol, synergistically exhibit whitening or moisturizing action, by dissolving idebenone, the first active ingredient, in a solvent containing the second active ingredient, bisabolol. Nanoliposomes inserted into the phospholipid bilayer were prepared.

Since the nanoliposome of the present invention has a complex in which idebenone is stably dissolved or dispersed in bisabolol, it is possible to transdermally deliver two active ingredients simultaneously with high efficiency.

In addition, since bisabolol has an antioxidant action by itself, it exhibits a synergistic effect with idebenone in pharmacological actions such as whitening or moisturizing, so that the nanoliposome of the present invention has an optimal stability of idebenone. And it can have an excellent cosmetic effect even including the active ingredient of bisabolol.

In addition, the phospholipid bilayer of the nano-liposome of the present invention can improve the transdermal delivery efficiency and stability of the nano-liposome by additionally containing a fatty acid and an anionic surfactant.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a schematic diagram of a nano-liposome of the present invention.
2 is a diagram showing the cell viability when idebenone and / or α-bisabolol is added.
Figure 3 is a diagram showing the results of measuring the melanin-producing ability when idebenone and / or α-bisabolol treated in the B16-F10 cell line.
4 is a diagram showing the results of measuring the expression level of genes involved in melanogenesis when idebenone and/or α-bisabolol was treated in the B16-F10 cell line.
5 is a diagram showing the value of ROS removal ability measured by the DCF-DA antioxidant experiment targeting idebenone and / or α-bisabolol.
6 and 7 are diagrams showing the results of confirming the degree of skin permeation of idebenone by treating the artificial skin with Examples 1, 2, and Comparative Example 1 in Experimental Example 2-1.
8 is a view showing the results of confirming the degree of skin permeation of idebenone in Experimental Example 2-2, Example 3, Comparative Example 1, and the non-liposomalized idebenone solution was treated on artificial skin.
9 is a view showing the results of confirming the degree of skin permeation of idebenone by treating the artificial skin with Examples 4 to 6 in Experimental Example 2-3.
10 is a view showing the results of confirming the degree of skin permeation of idebenone by treating the artificial skin with Example 6 and the idebenone solution in Experimental Examples 2-4.
11 is a view showing the results of particle size analysis of the liposome suspension of Example 6.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in several different forms, and thus is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

The terms used herein are used only to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprises" or "have" are intended to designate that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Experimental Example 1. Confirmation of synergistic effect of idebenone and α-bisabolol complex

The present inventors devised a cosmetic formulation using the active ingredient α-bisabolol, which is known to have an antioxidant effect, as a solvent of idebenone while searching for a solvent to maximize the skin penetration effect of idebenone, which is poorly soluble. .

First, in the complex in which idebenone is dissolved in α-bisabolol, it was attempted to determine whether the effect on target cells was increased due to the physical and chemical effects of the two components on each other.

Experimental Example 1-1: Cytotoxicity measurement

Idebenone and α-bisabolol were mixed by concentration, and cytotoxicity was confirmed by measuring cell viability compared to the control group.

500 μL of the B16-F10 cell line was dispensed into each well at a concentration ^{of 1×10 5} cells/mL in a 24-well plate and _{cultured in an incubator at 37° C. 5% CO 2} for 24 hours. Then, 10 μM and 20 μM idebenone alone , 0.001% α-bisabolol alone and 10 μM of a complex of idebenone and α-bisabolol, the cell lines were treated and cultured for 24 hours. After that, 20 μL of MTT solution dissolved in PBS buffer was added and reacted for 4 hours to confirm formazan formation. After completely removing the medium, 100 μL of solubilization solution was added to dissolve formazan, ELISA reader (Synergy HTX Multi -Mode Reader, Bio-tex, USA) was used to measure the absorbance at 570 nm. Relative cell viability was obtained when the viability of control cells cultured without idebenone and/or α-bisabolol was assumed to be 100%. The results are shown in FIG. 2 .

In the group treated with idebenone alone, it did not show significant cytotoxicity even at the levels of 10 uM and 20 uM, and showed no cytotoxicity at all at 0.001% concentration of α-bisabolol. However, in the IDE&α-bisabolol complex, as it was confirmed that the cell viability dropped to 50% at the 10uM level, the complex concentration was adjusted lower than this and the next experiment was carried out.

Experimental Example 1-2: Measurement of melanin production

To evaluate the melanogenesis inhibitory effect of idebenone alone, α-bisabolol alone, and their complexes on the B16-F10 cell line, 2 mL of each ^{cell line was added to a 6-well plate to 3X10 5 cells/well.} Incubated for 24 hours. Next, 5 μM of idebenone alone, 0.0005% of α-bisabolol, and their complexes were treated with 5 μM, and 1 mM arbutin was treated as a positive control. Here, 100 nM of α-MSH was treated to stimulate the cells for 72, and then melanin generated inside and outside the cells was measured. Extracellular melanin was measured at 405 nm by transferring 100 μL of the culture medium to a 96 well plate. Intracellular melanin formed in the cells was harvested by treating the cultured cells with 0.25% Trypsin-EDTA solution and then centrifuged (10,000 rpm, 4°C for 5 minutes), and then added to the cell pellet 1 N NaOH (10% DMSO) was added, and the amount of melanin production was measured in the same manner at 405 nm by dissolving intracellular melanin for 60 minutes at 80° C. The results are shown in FIG.

As a result of measuring extracellular melanin, idebenone and α-bisabolol complex did not inhibit melanin formation more than arbutin in idebenone alone and α-bisabolol alone, whereas idebenone and α-bisabolol complex formed more melanin than arbutin. was found to inhibit. The results of intracellular melanin also showed the same results, it was found that the synergistic effect appeared according to the formation of two complexes.

Experimental Example 1-3: Confirmation of gene expression involved in melanogenesis

In order to evaluate the inhibitory effect of idebenone alone, α-bisabolol alone, and their complex on the expression of genes involved in melanogenesis on the B16-F10 cell line, the cell line was placed in a 6-well plate with ^{2 mL of 3X10 5} cells/well. Each was added and incubated for 24 hours. Next, 5 μM concentration of idebenone alone, 0.0005% of α-bisabolol, and their complex were treated with 5 μM, treated with 1 mM α-MSH, a melanogenesis inducer, and cultured for 40 hours, and cells were harvested. . After synthesizing cDNA from intracellular mRNA, the expression levels of four genes TYR, TRP-1, DCT, and MITF involved in melanin production were measured. As a positive control, a cell line treated with 1 mM arbutin was prepared, and expression levels of four genes were measured in the same manner. The results are shown in FIG. 4 .

In the group treated with idebenone alone, it could not be confirmed that the expression levels of DCT and MITF were significantly reduced compared to the positive control group, and it was found that the expression levels of TYR and TRP-1 decreased compared to the positive control group.

In the complex treatment group of idebenone and α-bisabolol, it was found that the expression levels of all four genes (TYR, TRP-1, DCT, MITF) were significantly reduced compared to the positive control group or the idebenone alone treatment group.

In particular, considering that α-bisabolol did not reduce the expression level of four genes compared to the positive control group, melanogenesis gene expression could be predicted as α-bisabolol binds to idebenone to form a complex. It was found that it contributed to the reduction to a non-existent level.

Experimental Example 1-4: DCF-DA antioxidant experiment

The antioxidant capacity of the DCF-DA sample was measured by measuring the degree of fluorescence by reacting with ROS present in the cell. B16F10 cell line was treated ^{in 94-well at 5X10 4} cells/well, and then cultured for 24 hours. 10 μM of idebenone alone, 0.001% α-bisabolol alone, 5 μM of idebenone and α-bisabolol complex and DCF-DA probe were treated together, followed by reaction at 37° C. for 1 hour. After washing with PBS, 100 μl of free radical initiator was treated, and measurements were made at 480 nm (excitation) and 530 nm (emission) by time. The results are shown in FIG. 5 .

As a result of the antioxidant experiment, all three groups were measured to be lower than the control value, and it was found that the complex exhibited antioxidant ability at a similar level even though it contained half the content of the single group. That is, it was found that the idebenone and α-bisabolol complex effectively removed ROS generated in the cell and exhibited excellent antioxidant activity.

Preparation Example 1. Preparation of liposome suspension

Based on the results of Experimental Example 1, an idebenone liposome suspension was prepared using a complex in which the active ingredient, idebenone, was dissolved in α-bisabolol.

Specifically, a liposome suspension was prepared by the following process.

1) Preparation of idebenone dissolving part

After mixing α-bisabolol in idebenone at a ratio of 1:3 at RT (room temperature), it was dissolved at 500 rpm for 5 minutes. Thereafter, an alcohol such as ethanol and a polyhydric alcohol such as 1,2-Hexanediol were further mixed.

2) Preparation of lipid lysate

Phospholipids such as lecithin, fatty acids such as Oleic acid and MCT, and anionic surfactants such as Potassium Cetyl Phosphate were added and mixed in a solvent such as glycerin, and then heated to 80° C. and dissolved at 500 rpm for 5 minutes. It was then cooled to room temperature.

3) Mixing of idebenone dissolving part and lipid dissolving part

The idebenone dissolving part and the lipid dissolving part were mixed, stirred at 500 rpm for 10 minutes, and then circulated 3 times using a high pressure homogenizer to form and homogenize the liposome.

4) Preparation of liposome suspension

A suspension was prepared by adding water to the liposome in which the idebenone and α-bisabolol complex were encapsulated through step 3).

Experimental Example 2. Skin penetration test of liposome suspension

In vitro skin absorption test - Experimental method

In order to measure the transdermal absorption rate of the liposome suspension of the present invention, the in vitro skin absorption test guidelines (Ministry of Food and Drug Safety) were followed. The in vitro skin absorption test method confirms that the test substance passes through the skin, and it is a method of measuring the amount transferred to the solution reservoir inside the skin after a certain period of time by applying or applying the test substance to the skin surface. Multicellular animal skin or artificial skin may be used. In particular, when artificial skin is used, repeated measurement of the test substance is possible, and it is possible to study the exposure conditions without using experimental animals. The in vitro skin absorption test can compare skin absorption and permeation according to the composition of the material, so it can be used as a useful model for risk assessment due to skin absorption in the human body.

In this experimental example, using an artificial Membrane Strat-M for transdermal absorption test from Millipore (USA), it was installed in the Franz-Diffusion Cell and System (Perme gear, USA) and tested. The Franz-Diffusion Cell was maintained in a non-occluded condition in which the donor chamber was not sealed with a para film. Other conditions were adjusted as shown in Table 1.

artificial skin Strat-M Membrane Area of skin 1.326665cm ² Volume of sample solution 0.5ml Receptor medium PBS Volume of Receptor medium 12ml Temperature 37 °C Stirbar speed 500rpm Sampling Aliquot 1ml Sampling time 24h

Each test substance was mixed with a solvent, loaded into the donor chamber, and after a certain period of time, a sample (idebenone) in the membrane and reservoir was collected and the component was analyzed by HPLC to confirm the skin permeability of the test substance. In addition, the residual amount remaining on the skin was measured. In the case of continuous exposure so that calculation is impossible, the absorption rate was calculated using the permeability constant (Kp). The analysis conditions of HPLC are shown in Table 2 below.

HPLC analysis conditions wavelength 480 nm When the detection time is fast
Increase the water content in the mobile phase. column 4.6 mm, * 200 mm, 4 μm, C18 mobile phase 95% methanol + 5% water flow per minute 1 mL sample injection volume 25 μl

Experimental Example 2-1. Whether the percutaneous absorption is improved by the use of anionic surfactants

According to the process of Preparation Example 1, Examples 1 and 2 using the anionic surfactant and Comparative Examples 1,2,3 without using the anionic surfactant were prepared with the composition shown in Table 3 below.

Example 1
(with SDS) Example 2
(Added Amphisol K) Comparative Example 1
(No surfactant, α-bisabolol added) Comparative Example 2
(No surfactant added) Comparative Example 3
(no addition of α-bisabolol) ingredient content content content content content lipid lysate EDTA-2Na 0.03 0.03 0.03 0.03 0.03 Lecithin (phospholipon 90H) One One 4 4 One Sodium dodecyl sulfate One 0 0 0 0 Potassium Cetyl Phosphate 0 One 0 0 One MCT 2 2 0 0 2 Glycerin 10 10 75 75 10 Idebenone dissolving part Ethanol 10 10 0 0 10 Bisabolol 1.5 1.5 0 1.5 0 Idebenone 0.5 0.5 0.5 0.5 0.5 propanediol 0 0 7 7 7 1,2-Hexanediol 2 2 2 2 2 DI-Water To 100 To 100 To 100 To 100 To 100

According to the method of Experimental Example 2, Examples 1 and 2 and Comparative Examples 1 to 3 were treated on artificial skin at a concentration of 5,000 ppm, and the degree of skin permeation was measured after 4 hours. It was tested twice, and the results are shown in Tables 4 and 6 (first experiment), and Table 5 and 7 (second experiment).

1st experimental unit (mAU*s) Reservoir Membrane synthesis 5,000ppm 142.95 336.21 479.17 Comparative Example 1 135.95 408.93 544.88 Comparative Example 2 124.99 108.81 233.8 Comparative Example 3 199.60 244.08 443.69 Example 1 147.93 254.11 402.05 Example 2

2nd experimental unit (mAU*s) Reservoir Membrane synthesis 5,000ppm 120.24 359.57 479.82 Comparative Example 1 238.78 250.99 489.77 Example 1 169.53 290.60 460.14 Example 2

When a surfactant such as Potassium Cetyl Phosphate was not used, the ratio of the amount delivered to the Reservoir to the total dose was remarkably low (Comparative Example 2), and when the complex was not formed with the α-bisabolol solvent, dermal penetration It was confirmed that the ratio of the amount delivered to Membrane was significantly low because the degree was low (Comparative Example 3). On the other hand, in Examples 1 and 2 using a surfactant while forming a complex with α-bisabolol solvent, cell delivery and dermal layer permeability were high.

On the other hand, SLS, a representative anionic surfactant, is EWG green grade but is avoided in cosmetics, so it can be seen that even if potassium cetyl phosphate is used instead of SLS, it shows a similar transdermal absorption rate and can minimize irritation.

Experimental Example 2-2. Whether the percutaneous absorption is improved by the use of oleic acid

According to the process of Preparation Example 1, Example 3 containing oleic acid and Comparative Example 4 containing a large amount of glycerin instead of oleic acid were prepared with the composition shown in Table 6 below.

Comparative Example 4
Example 3 ingredient content content lipid melt EDTA-2Na 0.03 0.03 Lecithin (phospholipon 90H) One One Potassium Cetyl Phosphate One One Oleic acid 0 0.2 MCT 2 2 Glycerin 30 10 Idebenone dissolving part Ethanol 10 10 Bisabolol 1.5 1.5 Idebenone 0.5 0.5 1,2-Hexanediol 2 2 water DI-Water To 100 To 100

According to the method of Experimental Example 2, Example 3, Comparative Example 1, Comparative Example 4 and the non-liposome idebenone solution was treated on artificial skin at a concentration of 1,000 ppm, and the degree of skin permeation was measured after 70 hours. The results are shown in Table 7 and FIG. 8 below.

Tertiary Experimental Units (mAU*s) Reservoir Membrane synthesis 1000ppm 212.84 187.55 400.40 Comparative Example 1 171.33 183.56 354.89 Idebenone (0.1%) + Bisabolol (0.3%) + Butylene Glycol (99.6%) 434.20 265.68 699.89 Idebenone (0.1%) + Bisabolol (0.3%) + Ethanol (99.6%) 223.07 220.83 443.91 Idebenone (0.1%) + Bisabolol (0.3%) + Propanediol (99.6%) 201.00 220.90 421.90 Comparative Example 4 378.56 142.46 521.02 Example 3

As such, it was found that when liposomes were prepared using oleic acid, the amount remaining in the membrane was small and the amount permeated to the reservoir was increased. It is interpreted that oleic acid, an unsaturated fatty acid, gives elasticity to the liposome membrane and prevents oxidation of the liposome, thereby increasing the stability and transdermal permeability of the liposome.

Experimental Example 2-3. Whether the percutaneous absorption is improved by the phospholipid bilayer

According to the process of Preparation Example 1, the liposome suspensions of Examples 4 to 6 with different phospholipid bilayer compositions were prepared with the composition shown in Table 8 below.

Example 4 Example 5 Example 6 ingredient content content lipid melt EDTA-2Na 0.03 0.03 0.03 Lecithin (phospholipon 90H) 0.5 One 2 Potassium Cetyl Phosphate 0.5 One 2 Oleic acid 0.1 0.2 0.4 MCT One 2 4 Glycerin 5 10 20 Idebenone dissolving part Ethanol 5 10 20 Bisabolol 1.5 1.5 1.5 Idebenone 0.5 0.5 0.5 1,2-Hexanediol 2 2 2 water DI-Water To 100 To 100 To 100

According to the method of Experimental Example 1, Examples 4 to 6 were treated on artificial skin at a concentration of 5000 ppm, and the degree of skin permeation was measured after 70 hours. The results are shown in Table 9 and FIG. 9 below.

4th experimental unit (mAU*s) Reservoir Membrane synthesis 5000ppm 339.2925 109.4116 448.7041 Example 4 400.5162 178.7104 579.2266 Example 5 646.9277 387.4342 1,034.362 Example 6

As such, it was found that in all of Examples 4 to 6, the transdermal absorption rate was high. In particular, when Lecithin and Oleic acid were included in 2 and 0.4 wt% of the total liposome suspension, respectively, the transdermal permeability was significantly high, and the total content was high, so it was found that long-term stability was maintained.

Experimental Example 2-4. Hourly percutaneous permeation test results

According to the method of Experimental Example 2, Example 6 and the idebenone dissolving part were treated on artificial skin at a concentration of 5000 ppm, and the degree of skin permeation was measured for 24 hours. After the initial administration of the sample to the donor chamber, the entire amount of solvent in the Reservoir was recovered when 6 hours had elapsed, and then, when 6 hours had elapsed, the solvent in the Reservoir was completely recovered, and when the last 12 hours had elapsed, all of the solvent in the Reservoir was recovered. Thus, the idebenone content was measured. The results are shown in Table 10 and FIG. 10 below.

5th experimental unit (mAU*s) 5,000ppm Reservoir Membrane synthesis 6 hours 6 hours 12 hours Example 6 380.78 349.68 521.18 203.92 1,455.56 Idebenone (0.5%) + Bisabolol (99.5%) 258.31 239.28 335.56 613.4 1,446.55 Idebenone (0.5%) + Bisabolol (1.5%) + Butylene Glycol (98%) 187.48 163.22 194.48 366.12 911.3

As such, the transdermal absorption rate of the liposome suspension was higher than that using the idebenone dissolution portion. In addition, compared with the solution dissolved in α-bisabolol without forming liposomes, the residual amount in Membrane was significantly lower, and as the amount detected in the Reservoir increased, it was found that the transdermal permeability was more excellent. In addition, in Example 6, it was confirmed that the absorption continued at a constant level even after a long period of time.

Experimental Example 3. Measurement of zeta potential of liposomes

The zeta potential is the potential difference between the medium and the fixed fluid layer attached to the dispersed particles. The zeta potential is used as a major indicator of the stability of colloidal dispersions. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between similarly charged adjacent particles in the dispersion. Specifically, the stability according to the zeta potential can be expressed as shown in Table 11.

Zeta potential [mV] 0 to ± 5 fast coagulation or agglomeration ± 10 to ± 30 initial instability ± 30 to ± 40 medium stability ± 40 to ± 60 excellent stability ± 61 Excellent stability

Table 12 shows the results of measuring the zeta potential of Example 6.

Example 6 Time / s pH Temp / degC Potential (Signal)
/mV Potential (ZP)
/mV Conductivity
/ mS/cm 0.00 6.77 26.91 -2,188.78 -75.61 1.06 5.00 6.77 26.91 -2,184.42 -75.46 1.06 10.00 6.77 26.91 -2,180.11 -75.31 1.06 15.00 6.77 26.91 -2,178.73 -75.26 1.06 20.00 6.77 26.91 -2,172.71 -75.06 1.06 25.00 6.77 26.90 -2,174.31 -75.11 1.06 30.00 6.77 26.90 -2,172.03 -75.03 1.06 35.00 6.77 26.90 -2,172.54 -75.05 1.06 40.00 6.77 26.88 -2,181.22 -75.35 1.06 45.00 6.77 26.88 -2,178.94 -75.27 1.06 50.00 6.77 26.88 -2,177.29 -75.21 1.06 55.00 6.77 26.88 -2,173.08 -75.07 1.06

As described above, the liposome according to the present invention has an average zeta potential value of -75 mV, which is shown to have high dispersion stability.

Experimental Example 4. Particle size analysis of liposomes

The particle size of the liposome can affect the pharmacokinetics in the body. That is, if the particle size is too small, the drug may not be effective for the desired time because the body loses it rapidly, and if it is too large, the transdermal absorption rate may be inhibited.

The particle size of Example 6 was analyzed using Malvern's particle size analyzer Zetasizer Nano ZS. The particle size analyzer analyzes the particle size by calculating the Brownian motion of the nanoparticles as a formula, and the particle size is larger than that of the scanning electron microscope analysis. The results are shown in FIG. 11 .

As a result of particle size analysis, the average particle size (D50) in Example 6 was about 200 nm, and D95 was 350 nm, confirming that it had a size suitable for transdermal absorption and high uniformity.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (9)

a phospholipid bilayer comprising an anionic surfactant and a fatty acid; and
Complex comprising idebenone and bisabolol
Containing, nano liposomes.
The nanoliposome according to claim 1, wherein the complex is inserted into the phospholipid bilayer.
According to claim 1, wherein the complex is characterized in that the liquid idebenone is dissolved or dispersed in a solvent containing bisabolol, nano-liposomes.
The nano-liposome according to claim 1, wherein the phospholipid bilayer has a negatively charged site of the anionic surfactant and a carboxyl group of a fatty acid on the surface of the nano-liposome.
The method according to claim 4, wherein the anionic surfactant is potassium cetyl phosphate, sodium lauryl sulfate, sodium dodecyl sulfate, sodium laureth sulfate, And sodium deoxycholate (sodium deoxycholate) will at least one selected from the group consisting of, nano-liposomes.
5. The method of claim 4, wherein the fatty acid is elaidic acid, lauric acid, limistic acid, palmitic acid, stearic acid, linoleic acid, oleic acid, linolenic acid, arachidonic acid, and caprylic/capric triglyceride (MCT) One or more selected from the group consisting of, nano liposomes.
According to claim 4, wherein the nano-liposome, based on 1 part by weight of idebenone, phospholipids, anionic surfactants, and fatty acids, respectively, 1 to 10 parts by weight, 1 to 10 parts by weight, and 0.1 to 1.0 parts by weight comprising that is, nano liposomes.
A cosmetic composition for whitening or moisturizing comprising the nano-liposome of any one of claims 1 to 7 as an active ingredient.
providing an idebenone dissolving portion comprising idebenone and bisabolol;
providing a lipid lysate comprising phospholipids; and
Comprising the step of mixing the idebenone dissolving unit and the lipid dissolving unit,
The phospholipids include anionic surfactants and fatty acids,
Method for preparing nano liposome suspension.

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