Preparation method of sponge bone needles coated with effective ingredients for improving skin and diamonds, and diamond sponge bone needle cosmetic composition containing same

Abstract

The present invention provides a method for preparing sponge bone needles, sponge bone needles prepared according to the method, and a cosmetic composition comprising the same. The sponge bone needles can promote the transdermal absorption of active ingredients and fine diamonds that can improve the skin. Moreover, the cosmetic composition of sponge bone needles prepared according to the present invention has excellent transdermal penetration effect and can be used for preparing cosmetic compositions with excellent functions.

Description

Technical Field

[0001] The present invention relates to a method for preparing a needle-like structure for promoting transdermal absorption, a needle-like structure prepared according to the method, and a cosmetic composition containing the needle-like structure, which can promote the transdermal absorption of active ingredients for improving the skin. Background Art

[0002] Appearance is considered one of the criteria for measuring health, so the concern for skin health is increasing day by day. The cosmetics industry is paying more and more attention to environmentally friendly materials that are harmless to the human body and have a functional impact on the skin. In particular, synthetic surfactants, pigments, antioxidants, chemical preservatives, etc. contained in cosmetics can cause skin dryness, or may cause rashes, inflammation, skin diseases, and even skin cancer. Therefore, in cosmetics, natural substances are needed as the main raw materials to replace the harmful chemical components to the skin. Especially in the field of functional cosmetics that pay attention to the additional functional effects of cosmetics, the research on natural functional substances has attracted much attention.

[0003] Among these natural substances, spongy spicules (sponge spicules), also known as sponges, are primitive marine organisms. They have no muscles, nerves, or organs. There are more than about 5,000 species and they can be found at any ocean depth. Spicules are needle-like structures present in invertebrates and have a skeletal function, mainly composed of silicic acid and calcium carbonate. The fibrous skeleton of sponges has an absorption ability based on capillary action and has been applied to various fields such as medicine. For example, the spicules of sponges contain antibiotics for treating osteomyelitis and are used to treat and prevent diseases.

[0004] The above-mentioned spongy spicules derived from sponges have a needle-like structure and are particularly suitable for delivering active ingredients to the transdermal layer of the skin. Therefore, attempts have been made to use them in cosmetic compositions.

[0005] The prior patent document of the present invention is Korean Patent No. 10-2074214.

[0006] Based on the above background, the present inventors have invented an excellent preparation method including a micro-diamond coating and an amino modification step, which can apply the above-mentioned needle-like structure derived from sponges to cosmetic compositions.

[0007] Content of the Invention

[0008] Technical Problem to be Solved by the Invention

[0009] The technical problem to be solved by the present invention relates to a method for preparing spongy spicules with excellent transdermal absorption. More specifically, a method for preparing a needle-like structure and a needle-like structure prepared according to the method are provided. The needle-like structure contains active ingredients for improving the skin and is coated with diamond.

[0010] Another object of the present invention is to provide a cosmetic composition containing sponge bone needles prepared according to the method.

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and those skilled in the art to which the present invention pertains can clearly understand other unmentioned problems from the following description.

[0012] Technical means for solving the problem

[0013] To solve the above problems, the present invention provides a method for preparing sponge bone needles coated with an active ingredient for improving skin and diamond, comprising the following steps:

[0014] (a) Washing the sponge bone needles;

[0015] (b) Incorporating a first active ingredient into the inner pores of the washed sponge bone needles;

[0016] (c) Coating silica on the outside of the sponge bone needles having the first active ingredient incorporated in the inner pores;

[0017] (d) Reacting the silica-coated sponge bone needles with an aminosilane and lysine to coat amino groups; and

[0018] (e) Coating a second active ingredient and diamond on the amino-coated silica.

[0019] According to one aspect, the aminosilane in step (d) may be 3-aminopropyltrimethoxysilane.

[0020] According to one aspect, the lysine in step (d) may be poly-L-lysine.

[0021] According to one aspect, the weight ratio of the aminosilane and lysine in step (d) may be from 1:1 to 500:1.

[0022] According to one aspect, the diamond in step (e) may be at least one selected from the group consisting of microdiamonds, nanodiamonds, and colloidal diamonds.

[0023] According to one aspect, the first active ingredient and the second active ingredient may each be at least one selected from the group consisting of collagen, fish collagen, nano collagen, low molecular weight collagen, plant collagen, collagen peptide, amino acids, tripeptides, tetrapeptides, and water-soluble vitamins, and the first active ingredient and the second active ingredient may be the same or different ingredients.

[0024] According to another embodiment of the present invention, there is provided a cosmetic composition comprising sponge bone needles prepared by the method.

[0025] According to one aspect, the cosmetic composition has a skin exfoliating effect.

[0026] According to one aspect, the cosmetic composition has a wrinkle improving effect.

[0027] Effects of the Invention

[0028] According to the preparation method of the present invention, sponge bone needles with excellent amino modification degree can be obtained to promote transdermal absorption. Moreover, for the sponge bone needles prepared by the preparation method of the present invention, their inner pores can contain a large amount of hydrophilic active ingredients such as collagen, and their exteriors are coated with fine diamonds. Thus, excellent skin exfoliating and skin regeneration effects can be given, and transdermal absorption effect and synergistic effect can also be achieved, penetrating deep into the skin. Therefore, it can be used to prepare a cosmetic composition with excellent functionality.

[0029] It should be understood that the effects of the present invention are not limited to the above effects, but also include all effects deduced from the invention structure described in the detailed description and claims of the present invention. Description of the Drawings

[0030] Figure 1 The schematic diagram of the preparation method of the needle-like structure according to the preparation method of the present invention is shown.

[0031] Figure 2 The fluorescence chromaticity based on whether the inner pores contain fluorescent collagen is shown.

[0032] Figure 3 The fluorescence chromaticity of the preparation examples prepared by different preparation methods is shown.

[0033] Figure 4 The skin absorption degree of the cosmetic composition containing the needle-like structure of the preparation example is shown.

[0034] Figure 5 It is the evaluation of the skin exfoliating effect of the cosmetic composition containing the needle-like structure according to the preparation method of the present invention.

[0035] Figure 6 It is the evaluation of the eye wrinkle improving effect of the cosmetic composition containing the needle-like structure according to the preparation method of the present invention.

[0036] Figure 7 It is a graph showing the cell survival effect of the cosmetic composition containing modified sponge bone needles of the present invention.

[0037] Figure 8It is a graph showing the effect of the cosmetic composition containing modified sponge bone needles of the present invention on collagen synthesis in NHDF cells.

[0038] Figure 9 It is a photograph showing the effect of the cosmetic composition containing modified sponge bone needles of the present invention on the generation of collagen fibers in NHDF cells (green: collagen fibers, scale bar: 20 μm).

[0039] Figure 10 It is a graph showing the skin absorbency of Cosmetic Composition 3 containing modified sponge bone needles of the present invention and Cosmetic Composition 1 containing normal sponge bone needles.

[0040] Figure 11 It is a graph comparing the skin absorbency of Cosmetic Composition 3 containing modified sponge bone needles of the present invention and Cosmetic Composition 1 containing sponge bone needles without coated diamonds.

[0041] Figure 12 It is a graph showing the skin absorption depth of Cosmetic Composition 3 containing modified sponge bone needles of the present invention and Cosmetic Composition 1 containing sponge bone needles without coated diamonds.

[0042] Figure 13 It is a graph comparing the skin absorption depth of Cosmetic Composition 3 containing modified sponge bone needles of the present invention and Cosmetic Composition 1 containing sponge bone needles without coated diamonds.

[0043] Figure 14 It is a graph showing the skin absorption rate of Cosmetic Composition 3 containing modified sponge bone needles of the present invention and Cosmetic Composition 1 containing sponge bone needles without coated diamonds.

[0044] Figure 15 It is a graph comparing the skin absorption rate of Cosmetic Composition 3 containing modified sponge bone needles of the present invention and Cosmetic Composition 1 containing sponge bone needles without coated diamonds. Detailed Description

[0045] A method for preparing sponge bone needles according to an embodiment of the present invention relates to a method for preparing sponge bone needles containing effective ingredients for improving the skin and diamonds, and includes (a) a step of washing the sponge bone needles; (b) a step of encapsulating a first effective ingredient in the inner pores of the washed sponge bone needles; (c) a step of coating silica on the outside of the sponge bone needles having the first effective ingredient encapsulated in the inner pores; (d) a step of reacting the silica-coated sponge bone needles with an amino silane and lysine to coat amino groups; and (e) a step of coating a second effective ingredient and diamonds on the amino-coated silica.

[0046] The terms "sponge spicule", "needle-shaped body", or "needle-shaped structure" used in this specification refer to structures with a needle-shaped microstructure, and the "sponge spicule", "needle-shaped body", or "needle-shaped structure" described in this specification can be used interchangeably. Preferably, it has a needle-shaped structure derived from sponges, and more preferably, the sponge can be freshwater sponge (Spongilla lacustris). The spicules or spiculae of the sponge are used as the sponge spicules of the present invention.

[0047] The sponge spicule is composed of calcium or silica and may have a porous interior. The sponge spicule has a type I, Y-type, or X-type structure. Specifically, the sponge spicule can be of the monoaxon type, triaxon type, tetraxon type, or polyaxon type. And the triaxon type can be further divided into the triactine type or hexactine type. The "monoaxon" refers to a needle-shaped sponge spicule with a pointed tip. Preferably, the sponge spicule has a type I structure and a monoaxon type structure.

[0048] The sponge spicule is a product obtained by extracting from sponges and further purifying.

[0049] The coated sponge spicule may contain active ingredients for improving the skin inside. The sponge spicule of the present invention is characterized in that all natural impurities inside are removed and it contains pure sponge spicules. Therefore, active ingredients for improving the skin can be included in the internal porous structure of the sponge spicule.

[0050] The active ingredients for improving the skin can be cosmetically active ingredients, which may include ingredients for improving and enhancing skin function, ingredients for inhibiting the deterioration of skin conditions, or ingredients for improving skin defects or highlighting advantages. Non-limiting examples of the active ingredients include collagen, fish collagen, nano-collagen, low molecular weight collagen, plant collagen, collagen peptides, amino acids, tripeptides, tetrapeptides, and water-soluble vitamins, etc.

[0051] The term "collagen" used in this specification refers to a semi-crystalline aggregate of collagen molecules. Specifically, collagen is the main protein forming the extracellular matrix, which is composed of amino acids and forms a triple helix shape and is the main component of connective tissue. The collagen can be collagen derived from the human body. Collagen derived from the human body can be fibrillar collagen or non-fibrillar collagen.

[0052] The sponge bone needles can be sponge bone needles modified by surface coating. Specifically, diamonds can be coated on the surface. Compared with the sponge bone needles without diamond coating, the diamond-coated sponge bone needles can improve the skin absorption of the active ingredient, thereby increasing the skin absorption rate of the active ingredient.

[0053] The diamonds for coating can be diamond powder or liquid. The diamonds used here can be at least one selected from the group consisting of micro diamonds, nano diamonds, and colloidal diamonds. Specifically, the diamonds can be nano diamonds.

[0054] Moreover, the surface of the sponge bone needles can include a silica coating.

[0055] The silica coating refers to a coating with a substance containing silica.

[0056] Specific examples of the silica include tetraethylorthosilicate and siloxane polymer. The silica coating can modify the surface of the needle-shaped body, thereby improving the binding performance with the active ingredient.

[0057] The surface of the sponge bone needles can include an amino coating. The amino coating refers to a coating of a substance containing amino groups. The amino coating refers to coating a substance or amino acid with an amino group at the end on the surface of the sponge bone needles.

[0058] The amino coating can be an aminosilane coating and / or a lysine coating.

[0059] The term "amino silane" used in this specification is an N-silyl compound in which hydrogen is replaced by an amino group, and the chemical formula is SiH4. As a kind of silane coupling agent, the amino silane can form a hydrophilic coating.

[0060] The amino silane is N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, N-(vinylbenzyl-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride), N-(vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride, 3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, and their hydrolyzates, derivatives, or combinations.

[0061] As used in this specification, the term "lysine" as an amino acid may include polylysine, which is an amino acid polymer homologous to lysine. The lysine may be poly-L-lysine, poly-D-lysine, or a combination of both.

[0062] In a specific example, the amino silicic acid may be 3-aminopropyltrimethoxysilane. And the lysine may be poly-L-lysine.

[0063] The washing in step (a) is for removing impurities present in the inner hole of the needle-shaped body, which can be achieved with purified water and alkaline and acidic solutions. Preferably, in the washing process of the needle-shaped body, the needle-shaped body is first washed with purified water at least once, and then treated with acidic solutions such as sulfuric acid, hydrochloric acid, hydrofluoric acid, hydrogen peroxide, and alkaline solutions such as sodium hydroxide and potassium hydroxide.

[0064] According to one aspect, the inner hole inclusion in step (b) can be achieved by ultra-high pressure treatment with a pressure of 50 to 300 MPa for 1 to 10 minutes. The first active ingredient is preferably a hydrophilic active ingredient, and non-limiting examples thereof include amino acids, tripeptides, tetrapeptides, and water-soluble vitamins. Under such conditions, the active ingredient can be well included in the inner hole.

[0065] Preferably, the silica coating in step (c) is achieved by reacting tetraethylorthosilicate. The silica coating process can improve the binding performance with the active ingredient by modifying the surface of the needle-shaped body. And preferably, the coating reaction is carried out at a temperature of 25 to 50 °C for 30 minutes to 2 hours, and may include ultra-high pressure treatment with a pressure of 50 to 300 MPa for 1 to 10 minutes. Under such conditions, silica can be best coated.

[0066] According to one aspect, the amino silane in step (d) may be 3-aminopropyltrimethoxysilane, and the lysine in step (d) may be poly-L-lysine.

[0067] According to one aspect, in the step (d), the weight ratio of the aminosilane is higher than that of lysine. Specifically, the weight ratio of the aminosilane to lysine can be from 1:1 to 1000:1. Preferably, the weight ratio can be 500:1. The aminosilane reacts with the sponge bone needles in the form of a mixed solution with lysine. Preferably, different mixing ratios can be selected from 0.01 to 5 parts by weight relative to 1 part by weight of the sponge bone needles. If outside the above range, the inclusion efficiency of the sponge bone needles with diamonds and the active ingredients may be reduced.

[0068] As confirmed in Examples 5 and 6 below, when the mixed weight ratio of the aminosilane and lysine is 500:1, excellent fluorescence collagen inclusion ability and skin absorption degree are exhibited, and in particular, an excellent percutaneous absorption rate is exhibited.

[0069] The amino coating in the step (d) is a step of modifying the surface of the needle-shaped body, and preferably, it can be carried out after coating silica. Preferably, the reaction of coating the amino group is carried out at a temperature of 25 to 50 °C for 30 minutes to 2 hours, and a ultra-high pressure treatment of pressurizing at 50 to 300 MPa for 1 to 10 minutes is carried out. Under the reaction conditions, amino modification can be best achieved.

[0070] According to one aspect, the diamond in the step (e) can be at least one selected from the group consisting of micro diamonds, nano diamonds, and colloidal diamonds.

[0071] According to one aspect, the first active ingredient and the second active ingredient are each at least one selected from the group consisting of collagen, fish collagen, nano collagen, low molecular weight collagen, plant collagen, collagen peptide, amino acid, tripeptide, tetrapeptide, and water-soluble vitamins, and the first active ingredient and the second active ingredient can be the same or different ingredients.

[0072] According to another embodiment of the present invention, there is provided a cosmetic composition containing the sponge bone needles prepared by the above method. The content of the sponge bone needles is not particularly limited, and preferably, it contains 0.1 to 5 parts by weight based on 100 parts by weight of the cosmetic composition. Under the above composition, excellent percutaneous absorption rate and skin peeling effect are exhibited.

[0073] The active ingredients for improving the skin contained in the cosmetic composition may include ingredients for improving and enhancing skin functions, ingredients for inhibiting the deterioration of skin conditions, or ingredients for improving the drawbacks or highlighting the advantages of the skin, etc. There is no particular limitation on the content of the active ingredients. Preferably, based on 100 parts by weight of the cosmetic composition, it contains 0.05 to 1 part by weight. At the same time, the cosmetic composition according to the present invention, in addition to the active ingredients, also includes various ingredients commonly contained in cosmetic compositions. For example, it includes all substances commonly used in facial mask papers, such as dispersants, thickeners, surfactants, preservatives, moisturizers, stabilizers, solvents, pigments, and fragrances, etc.

[0074] According to one aspect, the cosmetic composition has a skin exfoliation effect.

[0075] According to one aspect, the cosmetic composition has a wrinkle improvement effect.

[0076] The cosmetic composition according to an embodiment of the present invention further includes purified water, antioxidants, stabilizers, pigments, or fragrances, etc., which are commonly used in the field of cosmetic compositions.

[0077] Moreover, the cosmetic composition according to an embodiment of the present invention can be prepared into common dosage forms. Specifically, the dosage forms that can be prepared include lotion (milk lotion), softening lotion, toner, astringent lotion, moisturizing lotion, milk moisturizing lotion, moisturizing body lotion, nourishing body lotion, massage cream, nourishing cream, moisturizing cream, eye cream, hand cream, foundation, essence, nourishing essence, eye essence, film, soap, facial cleansing foam, facial cleanser, facial cleansing cream, body lotion, body cream, body wash, suspension, gel, powder, paste, facial mask or facial mask sheet, or aerosol composition. And it can also be realized in various forms suitable for providing a sense of moisture to the skin, such as gel or cream, paste, solid, etc. Specifically, it can be a jelly paste (sherbet) or an ointment, etc., which is a gel cream type with a granular feeling. The compositions of the above dosage forms can be prepared according to methods well-known in the art.

[0078] According to a specific example, the composition further includes at least one selected from the group consisting of oils, purified water, emulsifiers, dispersants, pigments, fragrances, ultraviolet ray protectants, sweeteners, vitamins, and metal ion blockers. And according to an embodiment, the composition further includes 0.1 wt% to 3.0 wt% of a preservative.

[0079] Those skilled in the art can easily select the blending amounts of the additive ingredients such as the oil within the scope that does not damage the purpose and effects of the present invention.

[0080] According to a specific example, the cosmetic composition is preferably a cream. For example, when the cosmetic composition is a cream, it can uniformly contain the coated sponge spicules of the present invention, which can not only effectively moisturize but also effectively penetrate the sponge spicules into the skin.

[0081] According to a specific example, the cosmetic composition further contains additive ingredients commonly used in cosmetics, such as any common cosmetic ingredients selected from the group consisting of dispersants, thickeners, pigments, fragrances, fillers, preservatives, antiseptics, neutralizing agents, ultraviolet ray protectants, sweeteners, vitamins, free radical scavengers, metal ion blockers, and mixtures thereof.

[0082] Hereinafter, the embodiments will be described in detail with reference to the accompanying drawings. Various changes can be made to the embodiments, and the scope of this patent application is not limited to the above embodiments. It should be understood that all modifications, equivalents, and their substitutes are included within the scope of the claims.

[0083] The terms used in the embodiments are only for describing specific embodiments and are not intended to limit the scope of protection. Without special description in the content, singular expressions include plural meanings. In this specification, terms such as "including" or "having" are used to express the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and do not exclude the presence of one or more other features, numbers, steps, operations, components, parts, or combinations or additional functions thereof.

[0084] Without other definitions, all terms used herein, including technical or scientific terms, have the ordinary meanings understood by those of ordinary skill in the art. Commonly used terms such as those defined in the dictionary should be understood as having the meanings in the relevant technical content. Without a clear definition in this specification, they cannot be interpreted as idealized or overly formal meanings.

[0085] Moreover, in the process of description with reference to the accompanying drawings, regardless of the reference numerals, the same components are denoted by the same reference numerals, and repeated descriptions are omitted. In the process of describing the embodiments, when it is determined that a detailed description of related well-known technologies will unnecessarily confuse the embodiments, the detailed description thereof is omitted.

[0086] Example 1. Obtaining Sponge Spicules

[0087] Example 1-1. Washing the sponge spicules

[0088] The sponge spicules extracted from the Russian freshwater sponge L (Spongilla lacustris L) are used as needle-like structures, which are sponge structures with internal pores. The washing process of the sponge spicules with internal pores is as follows: 100 g of sponge spicules are put into 1000 ml of purified water to remove salts, filtered through a 400-mesh (mesh) polyethylene (PE) sieve, and the sponge spicules remaining on the sieve are repeatedly washed 2 times with purified water. Then, they are put into 1000 ml of 70% ethanol for sterilization, filtered through a 400-mesh polyethylene sieve, and the sponge spicules remaining on the sieve are repeatedly washed 2 times with purified water. Then, the washed sponge spicules are put into a dryer (Hanbaek Science Company) and dried at 50 °C.

[0089] To remove the impurities remaining on the dried sponge spicules, the dried sponge spicules are immersed in 1000 ml of 35% hydrogen peroxide (H2O2) (Samchon Chemical Company), and ultrasonically treated for 1 hour at 60 °C using an ultrasonic cleaner (Sungdong Ultrasonic Company) under the conditions of 300 W and 40 kHz. Then, after filtering through a 400-mesh polyethylene sieve, the sponge spicules remaining on the sieve are repeatedly washed 3 times with 500 ml of purified water. After that, the washed sponge spicules are put into a dryer and dried at 50 °C. The washing of the sponge spicules can be completed through the above process.

[0090] Example 1-2. Inclusion of the first active ingredient in the internal pores of the sponge spicules

[0091] Add 500 ml of 1N HCL (Sigma-Aldrich Company) to 100 g of the washed sponge spicules, then react for 1 hour, and then add 500 ml of 1N NaOH (Sigma-Aldrich Company) for neutralization. Then add 1000 ml of 10% sodium percarbonate and react for 1 hour to remove the impurities in the internal pores.

[0092] Put 10 g of the mixture containing sponge spicules and collagen as the first active ingredient into a plastic bag, vacuum-decompress it, and then use a high-pressure processor (Ilshin autoclave company, ) to pressurize it at a pressure of 150 MP for 5 minutes. Then, filter through a 400-mesh polyethylene sieve, and the sponge spicules remaining on the sieve are repeatedly washed 3 times with 500 ml of purified water. After that, the washed sponge spicules are put into a dryer and dried at 50 °C. The washing of the sponge spicules can be completed through the above process.

[0093] Example 1-3. Coating with silica

[0094] The sponge bone needles obtained from the above Examples 1-2 were placed in a 1% tetraethyl orthosilicate solution and reacted for 1 hour to coat the sponge bone needles with silica.

[0095] Example 1-4. Coating with Amino Groups

[0096] To 1 part by weight of the silica-coated sponge bone needles obtained from Examples 1-3, 0.01 part by weight of 3-aminopropyltrimethoxysilane, 0.01 part by weight of poly-L-lysine (mixing ratio 1:1), and 98.98 parts by weight of water were added and reacted for 1 hour to coat the sponge bone needles with amino groups. Then, the sponge bone needles were washed repeatedly 3 times with 500 ml of purified water. After that, collagen (from Sigma-Aldrich) conjugated with the fluorescent substance fluorescein-5-isothiocyanate (FITC, from Thermo Fisher Scientific) and 0.01 wt% of nanodiamonds were added and reacted at room temperature for 1 hour.

[0097] Then, the prepared mixture was filtered through a 400-mesh polyethylene sieve, and the diamond-binding structure remaining on the sieve was washed repeatedly 3 times with 500 ml of purified water. After that, the washed diamond-binding structure was placed in a dryer and dried at 50 °C. Through the above process, a needle-like structure with collagen in the inner pores and diamonds and fluorescent collagen bound to the amino groups of the sponge bone needles was obtained.

[0098] Example 2. Preparation Example with a Weight Ratio of Amino Silane to Poly-L-lysine of 10:1

[0099] Except for changing the composition of the solution for coating with amino groups, the preparation was carried out in the same manner as in Example 1. To 1 part by weight of sponge bone needles, 0.1 part by weight of 3-aminopropyltrimethoxysilane, 0.01 part by weight of poly-L-lysine (mixing ratio 1:1), and 98.89 parts by weight of water were added and reacted for 1 hour to coat with amino groups. Except for the composition of the solution for coating with amino groups, other reaction conditions were the same as in Example 1.

[0100] Example 3. Preparation Example with a Weight Ratio of Amino Silane to Polylysine of 100:1

[0101] Preparation was carried out in the same manner as in Example 1 except for changing the composition of the solution for coating amino groups. To 1 part by weight of sponge bone needles, 0.01 part by weight of 3-aminopropyltrimethoxysilane, 0.01 part by weight of poly-L-lysine (mixing ratio 1:1), and 97.99 parts by weight of water were added, and the reaction was carried out for 1 hour to coat amino groups. Except for the composition of the solution for coating amino groups, other reaction conditions were the same as in Example 1.

[0102] Example 4. Preparation Example with a Weight Ratio of Amino Silane to Poly-L-lysine of 500:1

[0103] Preparation was carried out in the same manner as in Example 1 except for changing the composition of the solution for coating amino groups. To 1 part by weight of sponge bone needles, 5 parts by weight of 3-aminopropyltrimethoxysilane, 0.01 part by weight of poly-L-lysine (mixing ratio 500:1), and 93.99 parts by weight of water were added, and the reaction was carried out for 1 hour to coat amino groups. Except for the composition of the solution for coating amino groups, other reaction conditions were the same as in Example 1.

[0104] Comparative Example 1

[0105] The sponge bone needles in which the first active ingredient was not included in the inner pores and which did not undergo the processes of coating silica and amino groups were washed 3 times repeatedly with 500 ml of purified water. Then, 0.01 wt% of collagen (Sigma-Aldrich) conjugated with the fluorescent substance FITC (Thermo Fisher Scientific) and nanodiamonds were added, and the reaction was carried out at room temperature for 1 hour. Then, the prepared mixture was filtered through a 400-mesh polyethylene sieve, and the diamond-binding structure remaining on the sieve was washed 3 times repeatedly with 500 ml of purified water. After that, the washed diamond-binding structure was placed in a dryer and dried at 50 °C. Through the above process, a needle-like structure with diamonds and collagen only on the outer surface of the sponge bone needles was obtained.

[0106] Comparative Example 2

[0107] Obtain silica-coated sponge bone needles obtained only through the processes of Examples 1-1 to 1-3. Then, wash the sponge bone needles repeatedly 3 times with 500 ml of purified water. After that, add 0.01 wt% of collagen conjugated with the fluorescent substance FITC (Sigma-Aldrich) and nanodiamonds, and react at room temperature for 1 hour. Then, filter the prepared mixture through a 400-mesh polyethylene sieve, and wash the diamond-binding structure remaining on the sieve repeatedly 3 times with 500 ml of purified water. After that, put the washed diamond-binding structure into a dryer and dry it at 50 °C. Through the above process, a structure in which diamonds and fluorescent collagen are bound to the amino group of the sponge bone needle can be obtained. The needle-like structure is not coated with an amino group.

[0108] Comparative Example 3

[0109] Add 0.01 parts by weight of a 3-aminopropyltrimethoxysilane solution and 99.9 parts by weight of water to 1 part by weight of silica-coated sponge bone needles obtained only through the processes of Examples 1-1 to 1-3, and react for 1 hour to coat with an amino group. Then, wash the sponge bone needles repeatedly 3 times with 500 ml of purified water. After that, add 0.01 wt% of collagen conjugated with the fluorescent substance FITC (Sigma-Aldrich) and nanodiamonds, and react at room temperature for 1 hour. Then, filter the prepared mixture through a 400-mesh polyethylene sieve, and wash the diamond-binding structure remaining on the sieve repeatedly 3 times with 500 ml of purified water. After that, put the washed diamond-binding structure into a dryer and dry it at 50 °C. Through the above process, a structure in which diamonds and fluorescent collagen are bound to the amino group of the sponge bone needle can be obtained. Poly-L-lysine is not used in the process of coating the amino group of the needle-like structure.

[0110] Table 1 summarizes the preparation conditions of the sponge bone needles in the comparative examples and examples and the results of the fusion rate of diamonds and collagen on the outside of the obtained sponge bone needle structures. The calculation formula for the diamond-collagen fusion rate (%) is as follows.

[0111] Fusion rate (%) = (fluorescence value of collagen bound to the sponge bone needle after fusion of diamond and collagen) / (fluorescence value of the total collagen used) x 100

[0112] Table 1

[0113]

[0114] As can be seen from Table 1, the diamond and collagen fusion rate of the examples of the present invention is excellent.

[0115] Experimental Example 1. Evaluating Inclusion in Inner Pores

[0116] To evaluate the inclusion of the inner pore, the sponge bone needles without inner pore inclusion of Comparative Example 1 and the sponge bone needles with inner pore inclusion of Comparative Example 2 were selected for evaluation. 5 mg of sponge and 1 mg of FITC were added to 0.2 ml of purified water to prepare a mixture. 0.1 m of sponge bone needles were taken from the prepared mixture and placed on a glass slide, and observed with a fluorescence microscope (Nikon, TS2-FL) using a GFP filter. The results are as Figure 2 shown. As Figure 2 shown, the sponge bone needles with inner pore inclusion of Comparative Example 2 showed excellent fluorescence compared to the sponge bone needles without inner pore inclusion of Comparative Example 1. That is, it was confirmed that the active ingredient could be impregnated into the interior of the sponge bone needles through the inclusion of the inner pore of the sponge bone needles.

[0117] Experimental Example 2. Evaluating Inclusion of Fluorescent Collagen

[0118] To evaluate the fluorescence collagen inclusion ability of the sponge bone needles, the sponge bone needles obtained in Comparative Examples 3 and 4 and Examples 1 to 4 were evaluated as follows: After 5 mg of sponge bone needles were put into 0.2 ml of purified water to prepare a mixture, 0.1 mg of sponge bone needles were taken from the prepared mixture and placed on a glass slide, and observed with a fluorescence microscope (Nikon, TS2-FL) using a GFP filter. The results are as Figure 3 shown. As Figure 3 shown, excellent inclusion ability of fluorescent collagen was confirmed in Example 4.

[0119] Experimental Example 3. Preparation of a Cosmetic Composition and Evaluation of Its Function

[0120] 3-1. Preparation of a cosmetic composition

[0121] Cosmetic compositions were prepared using the sponge bone needles of Comparative Example 2 and Examples 1 to 4 in Table 1. Specifically, 10.0 wt% glycerol (humectant), 3.0 wt% butylene glycol, 1.0 wt% sodium chloride, and 4.0 wt% hydrolyzed collagen were added to 59.675 wt% purified water, and stirred at a speed of 1,000 rpm for 10 minutes to prepare an aqueous lysate. Then, to prepare an oily lysate, 8.0 wt% triglyceride caprylate / caprate (oil), 4.0 wt% diisostearyl malate (oil), 4.0 wt% cetyl PEG / PPG-10 / 1 dimethicone (emulsifier), and 3.0 wt% butanediol dicaprylate / dicaprate were mixed and heated to dissolve at 60 °C, and the prepared aqueous lysate was added and stirred at a speed of 1,000 rpm to prepare an oily lysate. Then, 2 wt% was further added relative to the total weight of the cosmetic composition of the sponge bone needles (Comparative Examples 3 and 4 and Examples 1 to 4, respectively), and stirred at 2,000 rpm for 5 minutes at 60 °C. Finally, 0.025 wt% diamond powder, 1.0 wt% preservative, and 0.3 wt% fragrance were added, and stirred at 3,000 rpm for 5 minutes at 50 °C to prepare a cosmetic composition. Hereinafter, the cosmetic compositions prepared by formulating the sponge bone needles of Comparative Examples 3 and 4 are referred to as Composition 1 and Composition 2, respectively, and the cosmetic composition prepared by formulating the sponge bone needle of Example 4 is referred to as Composition 3.

[0122] 3-2. Evaluation of the skin absorption degree of the cosmetic composition

[0123] The following experiment was conducted to confirm the skin absorption (penetration) degree of Compositions 1 to 3 obtained in 3-1 above. Compositions 1 to 3 were respectively applied to porcine skin (Franz Cell Membrane, FCM, APURES), and the obtained porcine skin tissue sections were observed with a fluorescence microscope (Nikon, TS2-FL). The results are as Figure 4 and shown in Table 2. Here, the tissue sections were obtained through the following process: 0.25 g of the composition was evenly applied to porcine skin (2×2 cm) every 10 minutes and repeated 4 times, then left at room temperature for 24 hours, then placed in 2 ml of 3.7% formaldehyde (Sigma-Aldrich) for 20 minutes for stabilization, then washed 3 times with 1X phosphate buffered saline (PBS), then placed in an optimal cutting temperature compound (OCT, Sakura Finetech), and frozen at -20 °C, and then cut into a thickness of 15 μm with a cryostat microtome (Leica Biosystems).

[0124] Table 2

[0125] Differentiation Composition 1 Composition 2 Composition 3 Skin Penetration Rate (%) 0.2 0.5 22.3

[0126] Reference Figure 4 As shown in Table 2, no fluorescence was observed in the epidermis and dermis of the skin for Compositions 1 and 2, while fluorescence was observed in the epidermis and dermis of the skin for Composition 3. This indicates that the fluorescent protein penetrated into the epidermis and dermis, demonstrating that the cosmetic composition of the present invention has excellent skin absorption (penetration) degree.

[0127] 3 - 3. Evaluation of skin exfoliation

[0128] To determine the degree of skin horny layer exfoliation of the cosmetic compositions of Composition 1 and Composition 3, the horny layer exfoliation effects were tested on 15 adult men and women aged 20 to 40 years old, and the results are as Figure 5 shown. Specifically, the subjects pasted the Hilltop Chamber containing 10 wt% dihydroxyacetone on the inner side of the upper arm for 5 hours before applying the test sample. After 24 hours, the color of the colored part was measured with a colorimeter (Minolta Co., Ltd.) to measure the color difference before and after applying the test sample. An appropriate amount of the cosmetic compositions of Composition 1 and Composition 3 was applied to the affected area 2 times each in the morning and afternoon every day, and this process continued for 2 weeks. Then, the degree of decolorization was measured with a colorimeter to measure the time required to return to the original skin color. And the horny layer exfoliation rate was calculated using the following formula.

[0129] Horny layer exfoliation rate (%) = (Turnover time of blank - Turnover time when applying the test sample) / (Turnover time of blank) * 100 Turnover time of blank: The time required for the horny layer of the group not treated with the test sample to be replaced by a new horny layer

[0130] Turnover time when applying the test sample: The time required for the horny layer of the group treated with the test sample to be replaced by a new horny layer

[0131] As Figure 5 shown, the group treated with Composition 3 showed an excellent horny layer exfoliation rate.

[0132] 3 - 4. Evaluation of improvement of periorbital wrinkles

[0133] To confirm the skin anti - aging effect of Composition 3, according to the standard operating procedures (SOP) of the Cosmetic Clinical Research Support Center of Se - myeong University, it was evaluated whether the periorbital wrinkles were improved, and the results are as Figure 6As shown. Specifically, for 20 female subjects aged 20 - 55 years (average age 42.9 years), an appropriate amount of the cosmetic composition of Composition 3 was applied around the eyes twice a day, in the morning and afternoon, and this process continued for 4 weeks. To analyze the eye wrinkles, 2 weeks and 4 weeks later, the eye area of the subjects was imaged using Antera3D (Miravex Corporation, Ireland) under constant temperature and humidity conditions (22 ± 2°C, RH 40 - 60%), and the instrument analysis values were measured using the analysis program provided by the instrument (Antera3D software, Miravex Corporation, Ireland). Figure 6 The left - hand photo in the middle is a photo of the same area at week 0 and week 4, and the right - hand chart shows the reduction rate of eye wrinkles. With the use of the cosmetic composition of Composition 3, compared with week 0, the eye wrinkles at week 4 improved by 13.8%.

[0134] Experimental Example 4. Confirming the In vitro Collagen Generation Effect of the Cosmetic Composition

[0135] 4 - 1. Experimental Materials and Preparation

[0136] The ivory - colored cream of Composition 3 was used to confirm the in - vitro collagen production effect.

[0137] Fetal bovine serum (FBS), recombinant human fibroblast growth factor - B (rhFGF - B), insulin (INSULIN), gentamicin sulfate, amphotericin (GA1000), and fibroblast basal medium (FBM) used in cell experiments were purchased from Lonz Corporation (USA), and EZcytox was purchased from DAEILLAB SERVICE Company (Korea). Specifically, for the experiment to measure intracellular collagen production, media with final concentrations of 0.001%, 0.005%, and 0.010% of the cosmetic composition (hereinafter referred to as "experimental groups") were prepared. The following experiments were conducted at the PNK Skin Clinical Research Center.

[0138] 4 - 2. Selection of Cell Lines and Cell Culture

[0139] Normal Human Dermal Fibroblasts (NHDF), a human fibroblast cell line, was purchased from Lonz Corporation (USA) and cultured in an incubator at 37°C and 5% CO2 using an FBM medium containing 0.1% INSULIN, 0.1% rhFGF - B, 0.1% GA - 1000, and 2% FBS, and sub - cultured every 2 - 3 days.

[0140] 4-3. Evaluation of cell viability

[0141] The cultured NHDF cells were seeded into 24-well plates at a concentration of 2.0×10 4 cells / well and cultured for 24 hours. Then, the medium of each experimental group was replaced with serum-free fibroblast basal medium (serum-free FBM) and cultured for another 24 hours. After culturing, 50 μl of EZ-Cytox was added to each well and cultured for 1 hour. The cultured medium was transferred to a 96-well plate, and the absorbance was measured at 450 nm using a VERSA max microplate reader (Molecular device, LCC, USA). The average absorbance value of each sample group was calculated and compared with the absorbance value of composition 1 in the control group to investigate cell viability.

[0142] The results are as Figure 7 shown. It was confirmed that the cosmetic composition of the present invention has no cytotoxicity compared to composition 1 in the control group.

[0143] 4-4. Quantification of collagen production

[0144] The NHDF cells were seeded into 24-well plates at a concentration of 2.0×10 4 cells / well and cultured for 24 hours. Then, the medium of each experimental group was replaced with serum-free FBM and cultured for another 24 hours. After culturing for 24 hours, the supernatant was taken, and the amount of procollagen released in the medium was measured using a type I procollagen C-terminal propeptide (PIP) EIA kit. The absorbance was measured at 450 nm using a VERSA max microplate reader (Molecular device, LCC, USA).

[0145] The results are as Figure 8 shown. The production of intracellular collagen (PIP) in the cosmetic composition of the present invention is correlated with the concentration. Specifically, in the cosmetic composition with a concentration of 0.001% to 0.010%, the production of intracellular collagen (PIP) increased significantly from 138.52% to 166.20% in correlation with the concentration.

[0146] 4-5. Measurement of fluorescence images of collagen fiber formation

[0147] The NHDF cells were seeded into 24-well plates at a concentration of 2.0×10 4Cells were inoculated at a concentration of [X] cells / well into a 24-well plate and cultured for 24 hours. Then, the medium was replaced with serum-free FBM containing the test sample diluted at different concentrations, and the cells were cultured for another 24 hours. After 24 hours of culture, the cells were washed with HEPES-BSS, and then the intracellular collagen fibers were stained by immunofluorescence staining. Subsequently, the fluorescence expression level was measured using a fluorescence microscope. The results were expressed as mean ± standard deviation, and the experimental results were those of 3 or more independent experiments. Statistical significance was verified by independent sample t-test at a significance level of p < 0.05.

[0148] The results are as Figure 9 shown. It was confirmed that when treated with the cosmetic composition of the present invention, the production of collagen fibers can be promoted at all concentrations.

[0149] Experimental Example 5. Confirming the Skin Absorbability of the Cosmetic Composition

[0150] 5-1. Selection of experimental subjects

[0151] As shown in Table 3 below, (i) those with periorbital wrinkles, nasolabial folds, forehead wrinkles, neck wrinkles, and glabellar wrinkles were selected; (ii) the person in charge of the experiment or the person entrusted by the person in charge of the experiment fully explained the matters to be informed to the experimental subjects, and the experimental subjects voluntarily filled in and signed the consent form; (iii) they were in good health, without infectious skin diseases and chronic physical diseases; (iv) they could be followed up during the experiment; (v) 21 adult women aged 20 to 59 years old, and all of the above conditions had to be met.

[0152] Table 3

[0153]

[0154] The average age of the 21 experimental subjects was 48.762 years old, including 11 people aged 40-49 and 10 people aged 50-59. Among them, 14 had dry skin, 4 had combination dry skin, and 3 had normal skin.

[0155] In addition, in the medical history questionnaire regarding skin diseases, itching, stinging, erythema, cosmetic side effects, drug side effects, photosensitivity, and atopic diseases for each experimental subject, the experimental subjects had no such problems and no other medical history.

[0156] After explaining the experiment, the experimental subjects who agreed to participate in the experiment and signed the consent form were measured as shown in Table 4 below. After using the product, the skin symptoms were evaluated. After the experiment, an effectiveness and preference survey was conducted.

[0157] Table 4

[0158]

[0159] 5-2. Measurement method

[0160] For instrument evaluation, the experimental subjects were stabilized in a waiting room with constant temperature and humidity conditions of 20°C to 24°C in room temperature and 40 to 60% humidity for 30 minutes to adapt the skin surface temperature and humidity to the environment of the measurement space, and water intake was restricted during the stabilization period. For objective measurement, one researcher measured, and the same part was measured each time.

[0161] 5-3. Confirm skin absorbance, absorption depth, and absorption rate

[0162] Skin absorbance was measured using a 3D Raman microscope system 30 (manufactured by TOKYO INSTRUMENTS, Japan).

[0163] In particular, the 3D Raman system used in the experiment is a device that can achieve fine pitch adjustment in the x, y, and z directions, and can move the z-axis focal length from the skin surface to the skin interior in micrometers. Therefore, without damaging the skin of the experimental subjects, three-dimensional Raman spectroscopic images based on depth can be obtained, and the skin absorbance of cosmetics can be determined using these images.

[0164] After dividing two regions of 3×4 cm2 of experiment / control on the left forearm of the experimental subjects, the forearm was fixed on the device for three-dimensional Raman scanning, and measurements were taken before and 30 minutes after product use. The laser used for measurement was 785 nm, and the scanning step was fixed at 5 μm. Five key points were taken in the x and y axis directions, and eleven key points were taken in the z axis direction. The skin absorbance of the cosmetics could be measured to a depth of 50 μm from the skin surface, and could also be converted into absorption depth (μm) and absorption rate (μm / h).

[0165] On the 2D image, the z-axis was divided into 10 equal parts at 5-μm intervals, and a total of 10 stages were evaluated. Therefore, the skin absorption depth could be converted by multiplying the change in skin absorbance before and 30 minutes after product use by 5 μm, and the skin absorption rate could be converted by dividing the absorption depth by the use time of 0.5 hours.

[0166] As shown in Table 5, Figure 10 and Figure 11 it shows that 30 minutes after using Composition 3 of the present invention, the skin absorbance was significantly improved statistically compared to before use.

[0167] Moreover, Composition 3 showed a significant effect of 1.871 times and 187% compared to Composition 1 of the control group including sponge spicules without coated diamonds, and a statistically significant difference was confirmed even after 30 minutes of use. The above results indicate that the cosmetic composition of the present invention coated with diamonds has a significant skin absorption effect compared to the uncoated control group.

[0168] Table 5

[0169]

[0170] Improvement rate (%) = |(after - before)| / before * 100

[0171] #p<0.05 by Wilcoxon signed rank test

[0172] p<0.05 by Ranked ANCOVA

[0173] The results of skin absorption depth measurement are shown in Table 6, Figure 12 and Figure 13 as shown. Compared with before use, when using Composition 3 of the present invention, the skin absorption depth compared to the control group increased by 1.791 times and 179%. These results indicate that the cosmetic composition of the present invention coated with diamonds is absorbed deeper into the skin compared to the uncoated control group, with a significant effect.

[0174] Table 6

[0175]

[0176] #: p<0.05 by Wilcoxon signed rank test

[0177] The results of skin absorption rate measurement are shown in Table 7, Figure 14 and Figure 15 as shown. Compared with before use, when using Composition 3 of the present invention, the absorption rate increased by 1.971 times and 179% compared to the control group. This result indicates that the cosmetic composition of the present invention coated with diamonds can be absorbed by the skin faster compared to the uncoated control group, with a significant effect.

[0178] Table 7

[0179]

[0180] #: p<0.05 by Wilcoxon signed rank test

[0181] 5-4. Efficacy questionnaire survey

[0182] After using the experimental product, the experimental subjects were directly required to complete a questionnaire survey. The experimental items were divided into 5 scales, namely very good (4), good (3), average (2), poor (1), and very poor (0). The researchers calculated the percentage of the number of experimental subjects for each response to judge the effect of the experimental product.

[0183] As shown in Table 8 below, it was confirmed that the cosmetic composition of the present invention had a relatively high effect on improving the physical sensation.

[0184] Table 8

[0185]

[0186] *4: very good, 3: good, 2: average, 1: poor, 0: very poor

[0187] 5-5. Questionnaire survey evaluation of product preference

[0188] After using the experimental product, the experimental subjects were directly required to answer a questionnaire on the product usage experience. The evaluation items included skin moisture, skin smoothness, extensibility, fragrance, and overall usage experience. The evaluation was divided into 5 scales, namely very good (4), good (3), average (2), poor (1), and very poor (0).

[0189] As shown in Table 9 below, it was confirmed that the experimental subjects had a high preference for the cosmetic composition of the present invention.

[0190] Table 9

[0191]

[0192]

[0193] *4: very good, 3: good, 2: average, 1: poor, 0: very poor

[0194] 5-6. Safety and adverse reaction evaluation

[0195] The safety of the experimental product was comprehensively evaluated based on the abnormal reactions of all experimental subjects who used the experimental product and all the abnormal reactions reported during the experiment. The incidence of abnormal reactions was calculated as the safety evaluation data of the product. And during the experiment, a questionnaire was used to investigate whether abnormal skin symptoms occurred during the use of the experimental product and the degree of the symptoms. A comprehensive evaluation was made on the visual evaluation results of the researchers on the use site of the experimental product and the results of the experimental questionnaire survey. Thus, the stability and abnormal reactions were evaluated.

[0196] During the experiment, through the visual evaluation of the experimental site by the researchers, it was found that no special skin abnormal reactions occurred. Moreover, there were no reports related to skin abnormal reactions in the questionnaire survey results of the experimental subjects, nor were there any experimental subjects who had abnormal reactions during the experiment.

[0197] In summary, the embodiments have been described by way of limited embodiments and drawings, and those of ordinary skill in the art can make various changes and deformations based on the description. For example, the described technology is executed in a different order from the described method, and / or the components such as the described system, structure, device, circuit, etc. are combined or assembled in a different form from the described method, or are replaced or substituted by other components or equivalents, and appropriate results can also be obtained.

[0198] Therefore, other embodiments, other implementations, and equivalents of the claims all fall within the scope of the appended claims.

Claims

1. A preparation method of sponge bone needles coated with effective skin-improving ingredients and diamonds, characterized in that, it comprises the following steps: (a) Washing the sponge bone needles; (b) Incorporating a first active ingredient into the inner pores of the washed sponge bone needles; (c) Coating silica on the outside of the sponge bone needles having the first active ingredient incorporated in the inner pores; (d) Reacting the silica-coated sponge bone needles with an amino silane and polylysine to coat amino groups; and (e) Coating a second active ingredient and diamonds on the amino-coated silica, wherein the first active ingredient is a hydrophilic active ingredient, and the hydrophilic active ingredient is at least one selected from the group consisting of collagen, amino acids, tripeptides, tetrapeptides, and water-soluble vitamins, the second active ingredient is at least one selected from the group consisting of collagen, amino acids, tripeptides, and tetrapeptides, the first active ingredient and the second active ingredient are the same or different ingredients.

2. The preparation method of sponge bone needles according to claim 1, characterized in that, the collagen is at least one selected from the group consisting of fish collagen, nano collagen, low molecular weight collagen, plant collagen, and collagen peptides.

3. The preparation method of sponge bone needles according to claim 1, characterized in that, the amino silane in step (d) is 3-aminopropyltrimethoxysilane.

4. The preparation method of sponge bone needles according to claim 1, characterized in that, the polylysine in step (d) is poly-L-lysine.

5. The preparation method of sponge bone needles according to claim 1, characterized in that, the mixing ratio of the amino silane and polylysine in step (d) is 1:1 to 500:

1.

6. The preparation method of sponge bone needles according to claim 1, characterized in that, the diamond in step (e) is at least one selected from the group consisting of micro diamonds, nano diamonds, and colloidal diamonds.

7. A cosmetic composition, characterized in that, it contains sponge bone needles prepared by the method according to any one of claims 1 to 6.

8. The cosmetic composition according to claim 7, characterized in that, the cosmetic composition has a skin exfoliating effect.

9. The cosmetic composition according to claim 7, characterized in that, the cosmetic composition has a wrinkle-improving effect.

Images