KR20230150413A - Spicule coated with diamond and method for producing the same - Google Patents

Abstract

The present invention relates to a method for producing spicules capable of promoting the transdermal absorption of skin-improving active ingredients and fine diamonds, the spicules produced thereby, and a cosmetic composition containing the same. The spicule cosmetics manufactured according to the present invention The composition can have excellent transdermal penetration efficacy and can be used in the production of cosmetic compositions with excellent functionality.

Description

Diamond coated spicule and method for manufacturing same {SPICULE COATED WITH DIAMOND AND METHOD FOR PRODUCING THE SAME}

The present invention relates to modified spicules coated with diamond particles that can promote skin improvement, a cosmetic composition containing the same, and a method for manufacturing the same.

As appearance is recognized as a measure of health and interest in skin health increases accordingly, interest in eco-friendly materials in the cosmetics industry that are harmless to the human body but have a functional effect on the skin is increasing. In particular, synthetic surfactants, pigments, antioxidants, and chemical preservatives contained in cosmetics are known to have the potential to dry out the skin or cause rashes, inflammation, skin diseases, and even skin cancer. Instead of using chemical ingredients that are harmful to the skin, use natural ingredients instead. There is a high need for cosmetics that use substances as main ingredients. In particular, research on natural functional substances is receiving attention in the field of functional cosmetics, which focuses on the secondary functional effects of cosmetics.

Among these natural substances, spicules (sponge bone fragments) are also called sponges. Sponges are primitive marine organisms that do not have muscles, nerves, or organs. There are more than 5,000 species and they are found regardless of the depth of the sea. am. Bone fragments are needle-shaped tissues that exist in the body of invertebrates and are responsible for the function of the skeleton, and their main components are silicic acid and calcium carbonate. The fibrous skeleton of sponges has the ability to absorb through capillary action and is used in various fields, including medical applications. For example, bone fragments from sponges contain antibiotics for the treatment of osteomyelitis and are used for the treatment and prevention of the disease. Since the spicules derived from sponges described above have a needle-like structure, they are particularly suitable for delivering active ingredients to the transdermal layer of the skin, and attempts have been made to use them in cosmetic compositions.

KR 10-2074214 B1

Against this background, the present inventor has completed the invention and provided a spicule containing a fine diamond coating for using the above-described sponge-derived needle-like structure as a cosmetic composition.

The technical problem to be achieved by the present invention is to provide a method for manufacturing spicules with excellent transdermal absorption, and more specifically, to provide modified spicules containing skin-improving active ingredients and coated with diamonds.

Another object of the present invention is to provide a cosmetic composition containing the spicule.

One aspect of the present invention provides a modified spicule coated with diamond.

Another aspect of the present invention provides a cosmetic composition containing the modified spicule as an active ingredient.

Another aspect of the present invention provides a method for manufacturing spicules coated with ingredients effective for skin improvement and diamonds.

The spicule of the present invention may contain a large amount of hydrophilic active ingredients such as collagen in its inner pores, and is coated on the outside with fine diamonds to provide the excellent skin exfoliation and skin regeneration effects, and also has a synergistic effect with the transdermal absorption effect to penetrate the skin inside the skin. It penetrates deeply and can be used to manufacture cosmetic compositions with excellent functionality.

1 is a diagram showing a schematic diagram of the manufacturing method of the needle-shaped structure of the present invention.
Figure 2 is a photograph showing the degree of fluorescence of fluorescent collagen enclosed in the inner cavity of the spicule.
Figure 3 is a photograph showing the degree of fluorescence of spicules manufactured using different manufacturing methods.
Figure 4 is a photograph showing the degree to which the cosmetic composition prepared including the modified spicules is absorbed into the skin.
Figure 5 is an evaluation of the skin peeling effect of a cosmetic composition containing the modified spicule of the present invention.
Figure 6 is a photograph and graph evaluating the effect of improving wrinkles around the eyes of a cosmetic composition containing the modified spicule of the present invention.
Figure 7 is a graph showing the cell survival effect of a cosmetic composition containing the modified spicule of the present invention.
Figure 8 is a graph showing the collagen synthesis effect of the cosmetic composition containing the modified spicules of the present invention in NHDF cells.
Figure 9 is a photograph showing the effect of a cosmetic composition containing the modified spicules of the present invention on collagen fiber production in NHDF cells (green: collagen fibers, scale bar: 20 ㎛).
Figure 10 is a graph showing the left eye corner lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 11 is a graph showing the right eye corner lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 12 is a graph showing the left corner of the mouth lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 13 is a graph showing the right corner of the mouth lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 14 is a graph showing the forehead lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 15 is a graph showing the left nasolabial fold lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 16 is a graph showing the right nasolabial fold lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 17 is a graph showing the neck wrinkle lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 18 is a graph showing the forehead wrinkle lifting effect by the cosmetic composition containing the modified spicule of the present invention.
Figure 19 is a graph showing the effect of lifting deep glabellar wrinkles by a cosmetic composition containing modified spicules of the present invention.
Figure 20 is a graph showing the effect of lifting deep crow's feet wrinkles by a cosmetic composition containing the modified spicule of the present invention.
Figure 21 is a graph showing the effect of lifting deep nasolabial folds by a cosmetic composition containing the modified spicule of the present invention.
Figure 22 is a graph showing the effect of lifting deep eye wrinkles by a cosmetic composition containing the modified spicule of the present invention.
Figure 23 is a graph showing the effect of lifting fine wrinkles around the eyes by a cosmetic composition containing the modified spicule of the present invention.
Figure 24 is a graph showing the jowl area lifting effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 25 is a graph showing the effect of improving elasticity in the deepest nasolabial fold area by a cosmetic composition containing the modified spicule of the present invention.
Figure 26 is a graph showing the effect of improving elasticity in the deepest nasolabial fold area by a cosmetic composition containing the modified spicule of the present invention.
Figure 27 is a graph showing the effect of lifting the skin's outer elastic area by a cosmetic composition containing the modified spicule of the present invention.
Figure 28 shows the skin moisturizing effect by a cosmetic composition containing the modified spicule of the present invention.
Figure 29 is a graph showing the effect of improving skin density by a cosmetic composition containing the modified spicule of the present invention.
Figure 30 is a graph showing the skin absorption of a cosmetic composition containing modified spicules of the present invention (Nudique Dia Spitera Ampoule) and a cosmetic composition containing regular spicules (Nudique Spicule Ampoule).
Figure 31 is a graph comparing skin absorption of a cosmetic composition containing modified spicules of the present invention and a cosmetic composition containing spicules not coated with diamonds (control group).
Figure 32 is a graph showing the skin absorption depth of a cosmetic composition containing modified spicules of the present invention and a cosmetic composition containing spicules not coated with diamonds (control group).
Figure 33 is a graph comparing the skin absorption depth of a cosmetic composition containing modified spicules of the present invention and a cosmetic composition containing spicules not coated with diamonds (control group).
Figure 34 is a graph showing the skin absorption rate of a cosmetic composition containing modified spicules of the present invention and a cosmetic composition containing spicules not coated with diamonds (control group).
Figure 35 is a graph comparing the skin absorption rate of a cosmetic composition containing modified spicules of the present invention and a cosmetic composition containing spicules not coated with diamonds (control group).
Figure 36 is a graph showing the effect of improving neck wrinkles of a cosmetic composition containing the modified spicule of the present invention.
Figure 37 is a graph showing the effect of improving nasolabial folds of a cosmetic composition containing the modified spicule of the present invention.
Figure 38 is a graph showing the effect of improving wrinkles around the eyes of a cosmetic composition containing the modified spicule of the present invention.

One aspect of the present invention provides a modified spicule coated with diamond.

As used herein, the terms “spicule,” “acicular body,” or “acicular structure” refer to a needle-shaped microstructure that constitutes the skeleton of a sponge. The terms “spicule,” “acicular body,” and “acicular structure” may be used interchangeably.

As used herein, the term sponge is a simple metazoan belonging to the phylum Sponges. The sponge may be Spongilla lacustris .

The spicule is made of calcium or silica, and the spicule may have a porous interior. The spicule has an I-shaped structure, a Y-shaped structure, or an X-shaped structure. Specifically, the spicule may be Monoaxon-shaped, Triaxon-shaped, Tetraxon-shaped, or Polyaxon-shaped. Additionally, the Triaxon shape may be a Triactine or Hexactine shape. The “Monoaxon” refers to a needle-shaped spicule with a pointed end. Preferably, the spicule may have an I-shaped structure and a monoaxon shape.

The spicule may be obtained from a sponge and further purified.

The coated spicule may contain an active ingredient therein. The spicule of the present invention is characterized in that it contains pure spicules by removing all natural impurities therein. Therefore, the spicule may contain an active ingredient in its internal porous structure.

The active ingredient may be a cosmetically effective ingredient, and may include ingredients for improving or enhancing skin function, ingredients for suppressing deterioration of skin condition, or ingredients for complementing skin's shortcomings or highlighting its strengths. .

The spicule may include at least one active ingredient in a porous structure inside the spicule or on its surface. Additionally, there may be two or more active ingredients. Specifically, it may include a first active ingredient or a second active ingredient. Specifically, the first active ingredient may be included in the porous structure inside the spicule, and the second active ingredient may be coated on the surface.

The active ingredients include collagen, fish collagen, nanocollagen, low-molecular-weight collagen, vegetable collagen, hydrolyzed collagen, procollagen, atelocollagen, desamidocollagen, collagen peptides, amino acids, tripeptides, tetrapeptides, and water-soluble vitamins. It may be one or more selected from the group consisting of.

As used herein, the term “collagen” refers to a semi-crystalline aggregate of collagen molecules. Specifically, collagen is a major protein that forms the extracellular matrix formed of a triple helix composed of amino acids, and is the main component of connective tissue. The collagen may be human-derived collagen. Human-derived collagen may be fibrillar collagen or non-fibrillar collagen.

Specifically, the fibrillar collagen may be Type I collagen, Type II collagen, Type III collagen, Type V collagen, or Type XI collagen. In addition, the non-fibrillar collagen is Type IV collagen, Type IX collagen, Type XII collagen, Type XIV collagen, Type XIX collagen, Type XXI collagen, Type VIII collagen, Type VIII collagen, Type It could be collagen.

In one embodiment, the collagen may be Type I collagen, Type II collagen, Type III collagen, Type IV collagen, or Type V collagen.

The spicule may be modified by coating its surface. Specifically, the surface may be coated with diamond. These diamond-coated spicules can improve the skin absorption of the active ingredient compared to spicules that are not coated with diamonds, thereby increasing the skin absorption rate of the active ingredient.

The diamond used in the coating may be in the form of diamond powder or liquid form. The diamond used at this time may be one or more selected from the group consisting of microdiamonds, nanodiamonds, and colloidal diamonds. Specifically, the diamond may be a nanodiamond.

Additionally, the spicule may include a silica coating on its surface. The silica coating means coating with a material containing silicon dioxide.

One specific example of the silica may be tetraethylorthosilicate or siloxane polymer. The silica coating can improve the binding performance with the active ingredient by modifying the surface of the needle-shaped body.

The spicule may include an amino group coating on its surface. The amino group coating refers to a coating layer of a material containing an amino group. The amino group coating refers to coating the surface of the spicule with a material or amino acid containing an amino group at the terminal.

The amino group coating may be aminosilane coating and/or lysine coating.

As used herein, the term “amino silane” refers to an N-silyl compound in which hydrogen is replaced with an amino group, and its chemical formula is SiH4 and is also called aminosilane. The aminosilane is a type of silane coupling agent and may form a hydrophilic coating layer.

The aminosilane 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-aminopropyltriene Toxysilane may be a hydrolyzate thereof, a derivative thereof, or a combination thereof.

As used herein, the term “lysine” is a type of amino acid and may include polylysine, which is a homologous amino acid polymer of lysine. The lysine may be poly-L-lysine, poly-D-lysine, or a combination thereof.

In one embodiment, the aminosilane may be 3-aminopropyltrimethoxysilane. Additionally, the lysine may be poly-L-lysine.

In one embodiment, the aminosilane may be included in a higher weight than lysine. Specifically, the aminosilane and lysine may have a weight ratio of 1:1 to 1000:1. Specifically, the weight ratio is 1:1 to 900:1, 1:1 to 800:1, 1:1 to 700:1, 1:1 to 600:1, 10:1 to 900:1, 10:1 to 10:1. 800:1, 10:1 to 700:1, 10:1 to 600:1, 50:1 to 900:1, 50:1 to 800:1, 50:1 to 700:1, 50:1 to 600: 1, 100:1 to 900:1, 100:1 to 800:1, 100:1 to 700:1, 100:1 to 600:1, 200:1 to 900:1, 200:1 to 800:1, 200:1 to 700:1, 200:1 to 600:1, 300:1 to 900:1, 300:1 to 800:1, 300:1 to 700:1, 300:1 to 600:1, 400: It may be 1 to 900:1, 400:1 to 800:1, 400:1 to 700:1 or 400:1 to 600:1. Preferably, the weight ratio may be about 500:1.

The spicule may be additionally coated with collagen on its surface. At this time, the collagen is as described above.

In one embodiment, the diamond-coated modified spicule may include an amino group and a collagen coating. Additionally, the collagen coating and diamond coating on the surface of the spicule may be combined with amino groups on the surface of the spicule.

In one embodiment, the modified spicule has a silica coating from the surface; Amino group coating; It may be coated in the order of collagen coating and diamond coating.

Another aspect of the present invention provides a cosmetic composition containing the spicule as an active ingredient.

The spicule is the same as described above. Additionally, the cosmetic composition may be used for skin improvement, skin exfoliation, wrinkle improvement, elasticity enhancement, or skin moisturizing. Additionally, the spicule may be included in an amount of 0.1% to 10% by weight. Specifically, the spicule is 0.2% to 10% by weight, 0.3% to 10% by weight, 1% to 10% by weight, 1% to 5% by weight, 1% to 8% by weight, 1% by weight. It may contain from 5% to 5% by weight, from 0.5% to 3% by weight, or from 1% to 3% by weight.

The cosmetic composition may be a formulation commonly used in cosmetics. In one embodiment, the composition includes lotion (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, eye cream, hand cream, and foundation. , essence, nutritional essence, eye essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cream, body cleanser, suspension, gel, powder, paste, mask pack or sheet, or formulation containing an aerosol composition. It can be manufactured with In addition, it can be applied in various forms suitable for providing moisture to the skin, such as gel or cream, paste, or solid. Specifically, it may be a gel-cream form with a particle texture such as sherbet or slush. Compositions of this dosage form can be prepared according to methods conventional in the art.

In one embodiment, the composition may further include one or more selected from the group consisting of oils, purified water, emulsifiers, dispersants, colorants, fragrances, sunscreens, sweeteners, vitamins, and metal ion sequestrants. Additionally, in one embodiment, the composition may further include 0.1% to 3.0% by weight of a preservative. The mixing amount of additional components such as oils can be easily selected by a person skilled in the art within a range that does not impair the purpose and effect of the present invention.

The cosmetic composition according to one embodiment may preferably be in a cream formulation. For example, when the cosmetic composition has a cream formulation, it can evenly contain the coated spicules of the present invention, and can be effective in moisturizing the skin and in penetrating the spicules into the skin.

The cosmetic composition according to one embodiment also contains additional ingredients commonly used in cosmetics, such as dispersants, thickeners, pigments, flavors, fillers, preservatives, preservatives, neutralizers, sunscreens, sweeteners, vitamins, free-radical scavengers, It may further comprise any conventional cosmetic ingredients that may be selected from metal ion sequestering agents, and mixtures thereof.

Another aspect of the present invention is a method for manufacturing spicules,

(a) washing the spicule;

(b) enclosing the first active ingredient in the inner cavity of the cleaned spicule;

(c) coating the outside of the spicule containing the first active ingredient in the cavity with silica;

(d) reacting the silica-coated spicule with aminosilane and lysine to coat amino groups; and

(e) It provides a method for producing spicules containing diamonds and ingredients effective for skin improvement, including the step of coating the amino group-coated silica with a second active ingredient and diamonds.

Non-limiting examples of the active ingredients include collagen, fish collagen, nanocollagen, low-molecular-weight collagen, vegetable collagen, collagen peptides, amino acids, tripeptides, tetrapeptides, and water-soluble vitamins.

The cleaning in step (a) is to remove organic impurities present in the inner pores of the needle-shaped body, and can be performed using purified water and basic or acidic solutions. Preferably, the needle-type body is washed with purified water at least once and then treated with an acidic solution such as sulfuric acid, hydrochloric acid, hydrofluoric acid, or hydrogen peroxide and a basic solution such as sodium hydroxide or potassium hydroxide. You can.

According to one embodiment, the pore inclusion in step (b) may be accomplished by ultra-high pressure treatment in which a pressure of 50 to 300 MPa is applied for 1 to 10 minutes, and the first active ingredient is preferably a hydrophilic active ingredient. Non-limiting examples include amino acids, tripeptides, tetrapeptides, and water-soluble vitamins. Under the above conditions, the first active ingredient can be excellently incorporated into the inner cavity.

The coating of the silica in step (c) can preferably be achieved by reacting tetraethylorthosilicate. Additionally, the coating reaction is preferably carried out at 25 to 50°C for 30 minutes to 2 hours, and may include ultra-high pressure treatment in which a pressure of 50 to 300 MPa is applied for 1 to 10 minutes. Under the above conditions, silica coating can be achieved to the highest degree.

The aminosilane in step (d) may be 3-aminopropyltrimethoxysilane, and the lysine in step (d) may be poly-L-lysine. there is.

The mixing ratio of aminosilane and lysine in step (d) is as described above, and may preferably be in a ratio of 1:1 to 500:1. Aminosilane reacts with spicules in the form of a mixed solution with lysine, and the mixing ratio can preferably vary from 0.01 parts by weight to 5 parts by weight per 1 part by weight of spicules. If it is outside the above range, a problem may occur in which the efficiency of encapsulating the diamond and the active ingredient in the spicule is reduced.

As verified in Example 2 below, when the mixed weight ratio of aminosilane and lysine is about 500:1, it shows the best fluorescent collagen encapsulation ability and degree of skin absorption, and in particular, can show better transdermal absorption rate.

The coating of the amino group in step (d) is a step of modifying the surface of the needle-shaped body, and can preferably be performed after silica coating. The reaction for coating the amino group may preferably be performed at 25 to 50°C for 30 minutes to 2 hours, and ultra-high pressure treatment may be performed in which a pressure of 50 to 300 MPa is applied for 1 to 10 minutes. Under the above reaction conditions, excellent modification with amino groups can be achieved.

According to one embodiment, the diamond in step (e) may be one or more selected from the group consisting of microdiamonds, nanodiamonds, and colloidal diamonds.

According to one embodiment, the first active ingredient and the second active ingredient are each selected from the group consisting of collagen, fish collagen, nano collagen, low molecular weight collagen, vegetable collagen, collagen peptide, amino acid, tripeptide, tetrapeptide, and water-soluble vitamins. There is at least one active ingredient, and the first active ingredient and the second active ingredient may be the same or different ingredients.

Another aspect of the present invention provides a spicule manufactured by the above method.

In addition, another aspect of the present invention provides a cosmetic composition containing the spicule.

The content of the spicules is not particularly limited, but is preferably 0.1 to 5 parts by weight based on 100 parts by weight of the cosmetic composition. Specifically, the spicule is 0.2% to 10% by weight, 0.3% to 10% by weight, 1% to 10% by weight, 1% to 5% by weight, 1% to 8% by weight, 1% by weight. It may contain from 5% to 5% by weight, from 0.5% to 3% by weight, or from 1% to 3% by weight.

The above composition can show the best transdermal absorption rate and skin peeling effect.

The skin-improving active ingredients contained in the cosmetic composition may include ingredients for improving or improving skin function, ingredients for suppressing deterioration of skin condition, or ingredients for complementing skin's shortcomings or highlighting its strengths. The content of the active ingredient is not particularly limited, but is preferably 0.05 to 1 part by weight based on 100 parts by weight of the cosmetic composition. In addition, the cosmetic composition according to the present invention may contain various ingredients included in conventional cosmetic compositions in addition to the above useful ingredients. For example, it may include all substances commonly used in mask sheets, such as all dispersants, thickeners, surfactants, preservatives, moisturizers, stabilizers, solubilizers, pigments, and fragrances.

According to one embodiment, the cosmetic composition may have a skin exfoliation effect. Additionally, the cosmetic composition may have a wrinkle improvement effect.

The cosmetic composition according to an embodiment of the present invention may further include purified water, antioxidants, stabilizers, pigments, or fragrances commonly used in the cosmetic composition field.

Additionally, the cosmetic composition according to an embodiment of the present invention can be manufactured in a conventional formulation. Specifically, it can be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, emulsion foundation, wax foundation, or spray.

Hereinafter, embodiments will be described in detail with reference to the attached drawings.

However, various changes can be made to the embodiments, so the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

Example 1 Obtaining spicules

Example 1.1. Cleaning of spicules

Spicules extracted from Russian Spongilla lacustris L were used as needle-like structures. This is a spicule with an internal cavity formed. The spicule with the inner hole formed was cleaned through the following process. 100 g of spicules were placed in 1,000 ml of purified water to remove salts, filtered through a 400 mesh polyethylene (PE) strainer, and the spicules remaining in the strainer were washed twice with purified water. Afterwards, it was sterilized in 1,000 ml of 70% ethanol, filtered through a 400 mesh polyethylene (PE) strainer, and the spicules remaining in the strainer were washed twice with purified water. Afterwards, the washed spicules were placed in a dryer (Han Baek Science Co., Ltd.) and dried at 50°C.

To remove impurities remaining in the dried spicules, the dried spicules were placed in 1,000 ml of 35% hydrogen peroxide (H ₂ O ₂ ) (Samcheon Chemical Co., Ltd.) and washed at 60°C using an ultrasonic cleaner (Seongdong Ultrasonic Co., Ltd.) at 300 W. and treated with ultrasound for 1 hour under 40 kHz conditions. Next, the process of filtering through a 400 mesh polyethylene (PE) strainer and washing the spicules remaining in the strainer with 500 ml of purified water was repeated three times. Afterwards, the cleaned spicules were placed in a dryer and dried at 50°C to complete cleaning of the spicules.

Example 1.2. Inclusion of the first active ingredient in the inner cavity of the spicule

500 ml of 1N HCL (Sigma-Aldrich) was added to 100 g of the cleaned spicules, reacted for 1 hour, and then 500 ml of 1N NaOH (Sigma-Aldrich) was added to neutralize. Afterwards, 1,000 ml of 10% sodium percarbonate was added and reacted for 1 hour to remove organic impurities in the pores.

10 g of a mixed solution containing hydrolyzed collagen (Sigma Aldrich, USA) combined with spicules and the first active ingredient, fluorescent molecule (FITC), was placed in a plastic bag, vacuum-decompressed, and placed in a high-pressure processor (Ilshin Autoclave). , Suflux®) was pressurized at a pressure of 150 MPa for 5 minutes. Next, the process of filtering through a 400 mesh polyethylene (PE) strainer and washing the spicules remaining in the strainer with 500 ml of purified water was repeated three times. Afterwards, the washed spicules were placed in a dryer and dried at 50°C to complete cleaning of the enclosed spicules.

Example 1.3. silica coating

The spicules obtained in Example 1.2 were reacted in a 1% tetraethylorthosilicate solution for 1 hour to coat the spicules with silica.

Example 1.4. Amino group coating

Based on 1 part by weight of the silica-coated spicule obtained in Example 1.3, 0.0 l part by weight of 3-aminopropyltrimethoxysilane and 0.01 part by weight of poly-L-lysine (mixing ratio 1: 1) and 9.898 parts by weight of water were added and reacted for 1 hour to coat the spicules with amino groups. Afterwards, the process of washing the spicules with 500 ml of purified water was repeated three times. Afterwards, 0.01% by weight of collagen (Sigma-Aldrich) and nanodiamonds bound with the fluorescent material FITC (Fluorescein-5-isothiocyanate, ThermoFisher Scientific) were added and reacted at room temperature for 1 hour.

Next, the prepared mixed solution was filtered through a 400 mesh polyethylene (PE) strainer, and the diamond bond structure remaining in the strainer was washed with 500 ml of purified water, and the process was repeated three times. Afterwards, the washed diamond-bonded structure was placed in a dryer and dried at 50°C to obtain a needle-like structure containing collagen in the pores and diamond and fluorescent collagen bonded to the amino group of the spicule. However, the fluorescent collagen was used to confirm the content, and in Examples 4 to 8 below, collagen without fluorescence was used.

Example 2. Manufacturing example

Example 2.1. Preparation example of aminosilane to polylysine weight ratio of 10:1

It was prepared as in Example 1, except that the solution composition used for amino group coating was different. For 1 part by weight of spicule, 0.1 part by weight of 3-aminopropyltrimethoxysilane, 0.01 part by weight of poly-L-lysine (mixing ratio 10:1) and 9.889 parts by weight of water were added. Amino groups were coated by reacting for 1 hour. Except for the solution composition used for amino group coating, all other reaction conditions were the same as Example 1.

Example 2.2. Preparation example of aminosilane to polylysine weight ratio of 100:1

It was prepared as in Example 1, except that the solution composition used for amino group coating was different. For 1 part by weight of spicule, 1 part by weight of 3-aminopropyltrimethoxysilane, 0.01 part by weight of poly-L-lysine (mixing ratio 100:1), and 9.799 parts by weight of water were added and left for 1 hour. Amino groups were coated by reacting for a while. Except for the solution composition used for amino group coating, all other reaction conditions were the same as Example 1.

Example 2.3. Example preparation of aminosilane to polylysine weight ratio of 500:1

It was prepared as in Example 1, except that the solution composition used for amino group coating was different. For 1 part by weight of spicule, 5 parts by weight of 3-aminopropyltrimethoxysilane, 0.01 parts by weight of poly-L-lysine (mixing ratio 500:1) and 9.399 parts by weight of water were added and left for 1 hour. Amino groups were coated by reacting for a while. Except for the solution composition used for amino group coating, all other reaction conditions were the same as Example 1.

Example 2.4. Preparation of Comparative Example 1

The process of washing the spicule without encapsulating the first active ingredient in the spicule cavity and without silica coating or amino group coating was repeated three times with 500 ml of purified water. Afterwards, 0.01% by weight of collagen (Sigma-Aldrich) and nanodiamonds bound with the fluorescent material FITC (Fluorescein-5-isothiocyanate, ThermoFisher Scientific) were added and reacted at room temperature for 1 hour. Next, the prepared mixed solution was filtered through a 400 mesh polyethylene (PE) strainer, and the diamond bond structure remaining in the strainer was washed with 500 ml of purified water, and the process was repeated three times. Afterwards, the washed diamond-bonded structure was placed in a dryer and dried at 50°C to obtain a needle-like structure with diamond and collagen only on the outer surface of the spicule.

Example 2.5. Preparation of Comparative Example 2

A spicule coated only with silica was obtained through the processes of Examples 1.1 to 1.3. Next, the process of washing the spicules with 500 ml of purified water was repeated three times. Afterwards, 0.01% by weight of collagen (Sigma-Aldrich) and nanodiamonds bound to the fluorescent substance FITC were added and reacted at room temperature for 1 hour. Next, the prepared mixed solution was filtered through a 400 mesh polyethylene (PE) strainer, and the diamond bond structure remaining in the strainer was washed with 500 ml of purified water, repeating the process three times. Afterwards, the washed diamond-bonded structure was placed in a dryer and dried at 50°C to obtain a structure in which diamond and fluorescent collagen were bonded to the amino group of the spicule. This needle structure was not coated with amino groups.

Example 2.6. Preparation of Comparative Example 3

0.01 parts by weight of 3-aminopropyltrimethoxysilane solution and 9.99 parts by weight of water were added to 1 part by weight of spicules coated only with silica, obtained only through the processes of Examples 1.1 to 1.3, and reacted for 1 hour to produce amino groups. was coated, and then the process of washing the spicules with 500 ml of purified water was repeated three times. Afterwards, 0.01% by weight of collagen (Sigma-Aldrich) and nanodiamonds bound to the fluorescent substance FITC were added and reacted at room temperature for 1 hour. Next, the prepared mixed solution was filtered through a 400 mesh polyethylene (PE) strainer, and the diamond bond structure remaining in the strainer was washed with 500 ml of purified water, repeating the process three times. Afterwards, the washed diamond-bonded structure was placed in a dryer and dried at 50°C to obtain a structure in which diamond and fluorescent collagen were bonded to the amino group of the spicule. Poly-L-lysine was not used in the amino group coating of this needle-like structure.

The spicule manufacturing conditions of the comparative examples and examples and the results of the fusion rate of the external diamond and collagen of the resulting spicule structure are summarized in Table 1 below. The diamond collagen fusion rate (%) was calculated as follows.

[Equation 1]

Fusion rate (%) = (Fluorescence value of collagen bound to spicules after diamond collagen fusion)/(Fluorescence value of total collagen used) x 100

As a result, according to Table 1, it can be seen that the diamond and collagen fusion rate of the examples of the present invention is excellent.

Example 3. Inclusion evaluation

Example 3.1. Evaluation of internal cavity inclusions

For evaluation of internal cavity encapsulation, the spicules of Comparative Example 1 in which internal cavity encapsulation was not achieved and the spicules in Comparative Example 2 in which internal cavity inclusion was achieved were selected and evaluated. After preparing a mixed solution by mixing 5 mg of spicules and 1 mg of FITC in 0.2 ml of purified water, 0.1 mg of spicules were taken from the prepared mixture, placed on a glass slide, and observed with a GFP filter under a fluorescence microscope (Nikon, Ts2-FL). did.

As a result, as shown in FIG. 2, the spicule of Comparative Example 2 with internal hole inclusion showed superior fluorescence compared to the spicule of Comparative Example 1 without internal hole inclusion. In other words, it was confirmed that the active ingredient can be impregnated inside the spicule through encapsulation of the spicule's inner pores.

Example 3.2. Fluorescent collagen inclusion evaluation

To evaluate the spicule's ability to encapsulate fluorescent collagen, the spicules obtained in Comparative Examples 3 and 4 and Examples 1 to 2 were evaluated as follows. After mixing 5 mg of spicules in 0.2 ml of purified water to prepare a mixed solution, 0.1 mg of spicules was taken from the prepared mixed solution, placed on a glass slide, and observed with a fluorescence microscope (Nikon, Ts2-FL) GFP filter.

As a result, as shown in Figure 3, it was confirmed that the inclusion of fluorescent collagen in the spicules obtained in Example 2.3 was excellent.

Example 4. Preparation and functional evaluation of cosmetic composition

Experimental Example 4.1. Cosmetic composition manufacturing

A cosmetic composition was prepared using the spicules of Comparative Example 2 and Examples 1 to 2 of Table 1 above. Specifically, 10.0% by weight of glycerin (humectant), 3.0% by weight of butylene glycol, 1.0% by weight of sodium chloride, and 4.0% by weight of hydrolyzed collagen were added to 59.675% by weight of purified water and stirred at 1,000 rpm for 10 minutes to form an aqueous solution. was manufactured. Afterwards, to prepare an oily melt, 8.0% by weight of caprylic/capric triglyceride (oil), 4.0% by weight of diisostearyl malate (oil), and cetyl PEG/PPG-10/1 dimethicone ( 4.0% by weight of emulsifier and 3.0% by weight of butylene glycol dicaprylate/dicaprate were mixed and dissolved by heating at 60°C. The prepared aqueous solution was added and stirred at 1,000 rpm to prepare an oily solution.

Afterwards, 2% by weight of the spicules (Comparative Examples 3 and 4 and Examples 1 to 4, respectively) were added based on the total weight of the cosmetic composition and stirred at 2,000 rpm at 60°C for 5 minutes. Finally, a cosmetic composition was prepared by adding 0.025% by weight of diamond powder, 1.0% by weight of preservative, and 0.3% by weight of fragrance and stirring at 3,000 rpm at 50°C for 5 minutes.

Hereinafter, the cosmetic compositions obtained by mixing the spicules of Comparative Examples 3 and 4 will be referred to as Compositions 1 and 2, respectively, and the cosmetic compositions obtained by mixing the spicules of Example 2.3 will be referred to as Composition 3.

Example 4.2. Evaluation of skin absorption of cosmetic compositions

An experiment was conducted to confirm the degree of skin absorption (penetration) of compositions 1 to 3 obtained in above 4.1. Tissue sections of the pig skin obtained through the process of applying each of the compositions 1 to 3 to pig skin (Franz Cell Membrane, FCM, APURES) were observed with a fluorescence microscope (Nikon, Ts2-FL), and the results are shown in Figure 2. 4 and Table 2. At this time, the tissue section was prepared by spreading 0.25 g of the composition on pig skin (2 × 2 cm), repeating the process four times at 10-minute intervals, keeping it at room temperature for 24 hours, and then incubating with 37% formaldehyde (Sigma-Aldrich). G) Fix by placing in 2ml for 20 minutes, wash 3 times with 1 It was obtained by cutting it into thick pieces.

Referring to Figure 4 and Table 2, it was confirmed that compositions 1 and 2 showed no fluorescence in the dermis and epidermis layers of the skin, while composition 3 showed fluorescence in the skin epidermis and dermis layers. This is because the fluorescent protein penetrated into the epidermis and dermis layers, supporting the fact that the cosmetic composition according to the present invention has an excellent degree of skin absorption (penetration).

Example 4.3. Skin peeling assessment

In order to confirm the degree of skin exfoliation of the cosmetic compositions of Composition 1 and Composition 3, the exfoliation effect was measured on 15 adult men and women aged 20 to 40, and the results are shown in Figure 5. Specifically, before applying the sample, the test subject put 10% by weight of dihydroxyacetone in a hilltop chamber and attached it to the inside of the upper arm for 5 hours. After 24 hours, the color of the colored area was measured using a chromatometer (Minolta) to measure the difference in color before and after applying the sample. Appropriate amounts of the cosmetic compositions of Composition 1 and Composition 3 were taken and applied to the affected area twice every morning and afternoon for two weeks.

Afterwards, the degree of discoloration was measured with a chromatometer and the time taken to return to the original skin color was measured. The keratin exfoliation rate was calculated using the following equation.

[Equation 2]

Keratin exfoliation rate (%) = (Turnover time of blank - Turnover time when applying sample)/(Turnover time of blank)*100

Turnover time of blank: The time it takes for the stratum corneum of the untreated sample to be replaced by a new stratum corneum.

Turnover time when applying sample: The time it takes for the stratum corneum of the sample treatment group to be replaced by a new stratum corneum.

As a result, as shown in Figure 5, the group treated with Composition 3 showed an excellent exfoliation rate.

Example 4.4. Eye wrinkle improvement evaluation

In order to confirm the effectiveness of Composition 3 in preventing skin aging, the improvement of wrinkles around the eyes was evaluated based on the standard operating instructions (SOP) of the Semyung University Cosmetics Clinical Research Support Center, and the results are shown in Figure 6.

Specifically, 20 female test subjects aged 20 to 55 years (average age 42.9 years) took an appropriate amount of the cosmetic composition of Composition 3 and applied it to the eye area twice every morning and afternoon for 4 weeks.

Afterwards, to analyze wrinkles around the eyes, the test subject's eyes were imaged with Antera 3D (Antera 3D, Miravex, Ireland) after 2 and 4 weeks under constant temperature and humidity (22±2℃ RH40~60%) conditions, and then Instrumental analysis values were measured using an analysis program (Antera 3D software, Miravex, Ireland). The left photo in Figure 6 is a photo of the same area at week 0 and week 4, and the graph on the right shows the reduction rate of wrinkles around the eyes.

As a result, as shown in Figure 6, it was confirmed that the wrinkles around the eyes were improved by 138% at week 4 compared to week 0 by using the cosmetic composition of Composition 3.

Example 5. Confirmation of in vitro collagen production effect of cosmetic composition

Example 5.1. Experimental materials and preparation

The in vitro collagen production effect was confirmed using the ivory-colored cream-colored formulation of Composition 3.

Fetal bovine seru (FBS), r-Human Fibroblast Growth Factor-B (rhFGF-B), INSULIN, Gentamicin sulfate, Amphotericin- (GA-1000) Fibroblast Basal Medium (FBM) used in cell experiments were obtained from Lonz (USA). It was purchased and used, and EZ-cytox was purchased and used from Daeil Lab Service (Korea). In particular, for tests measuring intracellular collagen production, culture media with final concentrations of cosmetic compositions of 0.001%, 0.005%, and 0.010% (hereinafter “each experimental group”) were prepared. The following experiment was conducted at the PNK Skin Clinical Research Center.

Example 5.2. Cell line selection and cell culture

NHDF (Normal Human Dermal Fibroblasts) cells, a human fibroblast cell line, were purchased from Lonza and cultured using FBM medium containing 0.1% INSULIN, 0.1% rhFGF-B, 0.1% GA-1000, and 2% fetal bovine serum (FBS). Cultivation was carried out in a thermostat at 5% CO ₂ at ℃, and subculture was performed once every 2 to 3 days.

Example 5.3. Cell viability assessment

NHDF cells were inoculated into a 24-well plate at a concentration of 2.0×10 ⁴ cells/well and cultured for 24 hours. The medium for each experimental group was replaced with serum-free FBM and cultured for 24 hours. After incubation, 50 μl of EZ-Cytox was added per well and cultured for another 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 devices, LCC, USA). The average absorbance value for each sample group was obtained, and cell viability was investigated by comparing it with the absorbance value of the control group.

As a result, as shown in Figure 7, it was confirmed that the cosmetic composition of the present invention had no cytotoxicity compared to the control group.

Example 5.4. Quantification of collagen production

NHDF cells were inoculated into a 24-well plate at a concentration of 2.0×10 ⁴ cells/well and cultured for 24 hours. The medium for each experimental group was replaced with serum-free FBM and cultured for 24 hours. After 24 hours of incubation, the supernatant was taken and the amount of free procollagen in the medium was measured using the Procollagen Type I C-peptide (PIP) EIA Kit. Absorbance was measured at 450 nm using a VERSA max microplate reader (Molecular devices, LCC, USA). The positive control group was compared using Lascorbic acid (0.001 %).

As a result, as shown in Figure 8, the cosmetic composition of the present invention significantly increased the amount of intracellular collagen (PIP) production in a concentration-dependent manner. Specifically, the amount of intracellular collagen (PIP) produced was significantly increased from 138.52% to 166.20% in a concentration-dependent manner in the cosmetic composition at a concentration of 0.001% to 0.010%.

Example 5.5. Collagen fiber production fluorescence image measurement

NHDF cells were inoculated into a 24-well plate at a concentration of 2.0Х10 ⁴ cells/well and cultured for 24 hours, then exchanged with serum-free FBM containing samples diluted at each concentration and cultured for 24 hours. After 24 hours of incubation, the cells were washed with HEPES-BSS and intracellular collagen fibers were stained using immunofluorescence staining. Afterwards, the level of fluorescence expression was measured using a fluorescence microscope. All of the above results are expressed as mean value ± standard deviation, and the experimental results shown are the results of three or more independent experiments, and statistical significance was verified at the p < 0.05 significance level by independent t-test.

As a result, as shown in Figure 9, it was confirmed that when treated with the cosmetic composition of the present invention, the degree of collagen fiber production increased at all concentrations.

Example 6. Confirmation of lifting effect of cosmetic composition

Example 6.1. Selection of test subjects

As shown in Table 3 below, there are no exclusion conditions, (i) there are crow's feet, nasolabial folds, forehead wrinkles, neck wrinkles, and glabellar lines, and (ii) the test subject must be notified by the study director or a person authorized by the study director. After receiving a sufficient explanation of what to do, they voluntarily completed and signed the consent form, (iii) were healthy without infectious skin diseases or chronic physical diseases, (iv) were available for follow-up during the test period, and (v) were between 20 and 59 years of age. Twenty-one adult women aged 23 years were selected.

As shown in Table 4 below, the skin conditions of the 21 test subjects were 7 with dry skin, 6 with moderately dry skin, 6 with neutral skin, and 2 with normal skin.

In addition, as shown in Table 5 below, in the questionnaire regarding each test subject's experience with skin disease, itching, stinging, erythema, cosmetic side effects, drug side effects, photosensitivity, and atopic disease, there were no test subjects who were eligible and had no experience with other items. There were also no test subjects present.

The test subjects were instructed to use the test product after explaining how to use it. All test subjects visited a total of two times, and measurements were performed as shown in Table 6 below. After using the product, skin symptom evaluation was conducted. After the test was completed, a survey on effectiveness and preference was conducted.

Compliance is the percentage of the number of times used compared to the number of times it should be used during the test period. As shown in Table 7, the overall compliance rate of the 21 people who completed the test was 100.00%. Therefore, data from all test subjects were used to analyze the results.

Example 6.2. Use of cosmetic compositions

Composition 3, an off-white cream, was used, and the test product was stored at room temperature in a sealed state. The test product was provided by BN Co., Ltd., and a label indicating the test number, test subject number, product name, manufacturer, storage method, etc. was affixed for use by test subjects.

The test subject took an appropriate amount on a dry palm twice a week (Wednesday and Sunday) for a total of 4 weeks and applied it along the skin texture all over the dry face and neck. In addition, during the human application test period, the use of any cosmetics (e.g. functional cosmetics for whitening, wrinkle improvement, etc.) containing active ingredients that may affect the test results, other than the test products provided by this center, was prohibited.

Example 6.3. Measurement of lifting effect of cosmetic composition

For instrumental evaluation, the test subject rested for 30 minutes in a waiting room with constant temperature and humidity conditions of room temperature 20℃ to 2℃ and humidity 40 to 60% to adapt the skin surface temperature and humidity to the environment of the measurement space. While drunk, fluid intake was limited. For objective measurement, measurements were made by one researcher, and the same area was measured for each measurement.

Example 6.4. Check the eye tail lifting effect

To measure the eye tail lifting, the same left and right eye corner areas of the test subject were photographed at 30° left and right before and after using the test product using F-ray (BEYOUNG Co, Ltd, Korea). The captured image was analyzed using Image-Pro ^® plus (Media Cybernetics, USA) by drawing a parallel straight line from the corner of the eye and measuring the angle with the curve spanning the end of the eye corner. The analysis parameter is the angle, and as the angle value increases, it means that it is effective in improving eyelid lifting.

As a result, as shown in Table 8 and Figure 10, the parameters of the left corner of the eye increased by 2.567% immediately after use and by 5.216% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

As a result, as shown in Table 9 and Figure 11, the parameters of the right corner of the eye increased by 2.064% immediately after use and by 4.280% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.5. Check the mouth corner lifting effect

To measure mouth corner lifting, the same left and right corner of the mouth of the test subject was photographed at 30° left and right before and after using the test product using F-ray (BEYOUNG Co, Ltd, Korea). The captured images were analyzed using Image-Pro ^® plus (Media Cybernetics, USA) by drawing a parallel straight line from the line where the two lips meet and measuring the angle with the curve at the end of the mouth. The analysis parameter is the angle, which means that as the angle increases, there is a mouth corner lifting effect.

As a result, as shown in Table 10 and Figure 12, the parameters on the left side of the corner of the mouth increased by 2.883% immediately after use and by 5.770% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

As a result, as shown in Table 11 and Figure 13, the parameter on the right side of the corner of the mouth increased by 3.226% immediately after use and by 5.674% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.6. Check the forehead lifting effect

Forehead lifting was measured in the same central area of the forehead of the test subject before and after using the test product using the Depression-medium mode of Antera 3D CS (Miravex Ltd, Ireland), a 3D skin imaging device. The analysis parameter is the removed skin volume (㎣), and as the volume (㎣) value decreases, it means that there is a forehead lifting effect.

As a result, as shown in Table 12 and Figure 14, forehead wrinkles increased by 21.626% immediately after use and by 49.593% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.7. Confirmation of lifting effect in nasolabial fold area

The nasolabial fold area was measured using the Depression-medium mode of Antera 3D CS (Miravex Ltd, Ireland), a 3D skin imaging device, on the same left and right nasolabial fold areas of test subjects before and after using the test product. The analysis parameter is the removed skin volume (㎣), and as the volume (㎣) value decreases, it means that there is a lifting effect in the nasolabial fold area.

As a result, as shown in Table 13 and Figure 15, the left side of the nasolabial fold increased by 14.277% immediately after use and by 23.396% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

As a result, as shown in Table 14 and Figure 16, the right side of the nasolabial fold increased by 13.097% immediately after use and by 20.968% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.8. Check the lifting effect around the neck

The lifting of the neck wrinkle area was measured using the Depression-medium mode of Antera 3D CS (Miravex Ltd, Ireland), a 3D skin imaging device, in the same central area of the neck of the test subject before and after using the test product. The analysis parameter is the removed skin volume (㎣), and as the volume (㎣) value decreases, it means that there is a lifting effect in the neck wrinkle area.

As a result, as shown in Table 15 and Figure 17, compared to before use, neck wrinkles increased by 8.988% immediately after use and by 13.518% after 4 weeks of use, showing the effect of improving wrinkles and elasticity.

Example 6.9. Confirm the effect of alleviating deep forehead wrinkles

Deep forehead wrinkles were measured in the same central area of the forehead of the test subject before and after using the test product using the Wrinkle-medium mode of Antera 3D CS (Miravex Ltd., Ireland), a 3D skin imaging device. The analysis parameter is Maximum depth, which represents the maximum depth (mm). As the Maximum depth (mm) value decreases, it means that it is more effective in improving deep forehead wrinkles.

As a result, as shown in Table 16 and Figure 18, deep forehead wrinkles increased by 42.963% immediately after use and by 49.630% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.10. Confirm the effect of relieving deep eyebrow wrinkles

Deep glabellar wrinkles were measured in the same central area between the eyebrows of test subjects before and after using the test product using the Wrinkle-medium mode of Antera 3D CS (Miravex Ltd., Ireland), a 3D skin imaging device. The analysis parameter is Maximum depth, which indicates the maximum depth (mm). As the Maximum depth (mm) value decreases, it means that it is effective in improving deep glabellar wrinkles.

As a result, as shown in Table 17 and Figure 19, deep glabellar wrinkles increased by 9.155% immediately after use and by 11.972% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.11. Confirm the effect of relieving deep eye wrinkles

Deep crow's feet were measured using the Wrinkle-medium mode of Antera 3D CS (Miravex Ltd., Ireland), a 3D skin imaging device, on the same left crow's feet area of the test subject before and after using the test product. The analysis parameter is Maximum depth, which indicates the maximum depth (mm). As the Maximum depth (mm) value decreases, it means that it is effective in improving deep wrinkles at the corners of the eyes.

As a result, as shown in Table 18 and Figure 20, compared to before use, deep wrinkles at the corners of the eyes increased by 4.369% immediately after use and by 5.340% after 4 weeks of use, showing the effect of improving wrinkles and elasticity.

Example 6.12. Confirm the effect of relieving deep nasolabial folds

Deep nasolabial folds were measured in the wrinkle-medium mode of Antera 3D CS (Miravex Ltd., Ireland), a 3D skin imaging device, on the same left nasolabial area of the test subject before and after using the test product. The analysis parameter is Maximum depth, which represents the maximum depth (mm). As the Maximum depth (mm) value decreases, it means that it is more effective in improving deep nasolabial folds.

As a result, as shown in Table 19 and Figure 21, deep nasolabial folds increased by 11.111% immediately after use and by 15.033% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.13. Confirm the effect of relieving deep wrinkles around the eyes

Deep wrinkles around the eyes were measured in the same area around the left eye of the test subject before and after using the test product using the Wrinkle-medium mode of Antera 3D CS (Miravex Ltd., Ireland), a 3D skin imaging device. The analysis parameter is Maximum depth, which indicates the maximum depth (mm). As the Maximum depth (mm) value decreases, it means that it is effective in improving deep wrinkles around the eyes.

As a result, as shown in Table 20 and Figure 22, deep wrinkles around the eyes increased by 6.466% immediately after use and by 12.096% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.14. Confirm the effect of relieving fine wrinkles around the eyes

Fine wrinkles around the eyes were measured using PRIMOS CR Small Field (Canfield Imaging Systems, USA) on the same left eye area of the test subject before and after using the test product. The analysis parameter is the average roughness (Ra (㎛)), and the lower the Ra (㎛) value, the more effective it is in improving fine wrinkles around the eyes.

As a result, as shown in Table 21 and Figure 23, fine wrinkles around the eyes were improved by 5.412% immediately after use and by 5.850% after 4 weeks of use compared to before use.

Example 6.15. Check the effect of lifting the chin area

To measure the lifting of the jowl area, the jowl area viewed from the same right side of the test subject before and after using the test product was photographed using Vectra XT (Canfield Imaging Systems, USA), which can measure skin volume through 3D images. The analysis parameter is the jowl area volume (㏄), and the lower the volume (㏄) value, the more effective it is in improving the jowl area lifting.

As a result, as shown in Table 22 and Figure 24, the jowl area increased by 13.165% immediately after use and by 17.462% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.16. Confirm the effect of improving elasticity in the deepest nasolabial folds

The nasolabial area on the same side of the test subject was measured before and after using the test product using a Dermal Torque Meter (DTM) (DIA STRON, UK). The parameter is Ur/Ue, and as the Ur/Ue value increases, it means that it is effective in improving elasticity in the deepest nasolabial fold area.

As a result, as shown in Table 23 and Figure 25, the elasticity of the deepest nasolabial fold area increased by 15.198% immediately after use and by 12.775% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Additionally, the elasticity of the deepest nasolabial fold area was measured using Antera 3D CS. Specifically, the same left nasolabial area of the test subject was measured before and after using the test product using the Wrinkle-Large mode of Antera3D CS (Miravex Ltd., Ireland), a 3D skin imaging device. The analysis parameter is Maximum depth, which represents the maximum depth (mm). As the Maximum depth (mm) value decreases, it means that it is effective in improving elasticity in the deepest nasolabial fold area.

As a result, as shown in Table 24 and Figure 26, the elasticity of the deepest nasolabial fold area increased by 11.765% immediately after use and by 16.544% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.17. Confirm the effect of improving skin elasticity

Skin elasticity was measured on the same right cheek area of the test subject before and after using the test product using Cutometer ^® MPA580 (Courage+Khazaka electronic GmbH, Germany). The parameter is R2 (reformability of the skin), and as the R2 value increases, it means that it is effective in improving skin elasticity.

As a result, as shown in Table 25 and Figure 27, skin surface elasticity increased by 13.934% immediately after use and 7.049% after 4 weeks of use compared to before use, showing the effect of improving wrinkles and elasticity.

Example 6.18. Check the skin moisturizing effect

Skin moisture was measured on the same right cheek area of the test subject before and after using the test product using Corneometer ^® CM825 (Courage+Khazaka eletronic GmbH, Germany). The parameter is AU, and as the AU value increases, it means that it is effective in improving skin moisturization.

As a result, as shown in Table 26 and Figure 28, the skin moisturizing effect increased by 6.835% immediately after use and by 12.139% after 4 weeks of use compared to before use, resulting in a significant improvement in skin moisturizing ability.

Example 6.19. Skin density assessment

Skin density was measured on the same left cheek area of the test subject before and after using the test product using Skin Scanner DUB-USB (TPM taberna pro medicum, Germany). The parameter is Density (%), and as the % value increases, it means that it is more effective in improving skin density.

As a result, as shown in Table 27 and Figure 29, the effect of improving skin density was significantly improved, increasing by 11.774% after 4 weeks of use compared to before use.

Example 6.20. Validity Survey

After using the test product, participants were asked to directly answer the survey data in five levels: very good (4), good (3), average (2), bad (1), and very bad (0). The researcher determined the efficacy of the test product by calculating the percentage of the number of test subjects for each answer.

The measurement items are helpful in improving the lifting of saggy eye corners after using the test product, helping in improving the lifting of the corners of the mouth, helping in improving the lifting of sagging forehead, helping in improving the lifting of nasolabial folds, helping in improving the lifting of neck wrinkles, and helping in improving the deep forehead wrinkles that have already formed. Helps, helps improve already formed deep glabellar wrinkles, helps improve already formed deep crow's feet wrinkles, helps improve already formed deep nasolabial folds, helps improve already formed deep wrinkles around the eyes and fine wrinkles around the eyes that are not visible to the naked eye, It helps improve the lifting of sagging jowls, helps improve elasticity in the deepest nasolabial folds, helps improve skin (outer) elasticity, helps improve skin moisturization, and helps improve skin density.

As a result, as shown in Table 28 below, the average, above average, and percentage of the number of test subjects for the answers were derived for each item.

Example 6.21. Product preference survey

After using the test product, test subjects were asked to directly answer questionnaires about their feelings about using the product. The evaluation items are skin moisture, skin smoothness, applicability, absorption, fragrance, and overall feeling of use, with 5 levels: very good (4), good (3), average (2), bad (1), and very bad (0). It was evaluated as .

As a result, as shown in Table 29 below, the average for each questionnaire and the percentage of the number of test subjects who answered above average were derived.

Example 6.22. Safety and adverse reaction evaluation

The safety of the test product was determined by calculating the incidence rate of adverse reactions by combining all adverse reactions identified in all test subjects who used the test product and all adverse reactions reported during the test period, and used as product safety evaluation data. In addition, for abnormal skin symptoms that occurred while using the test product, the occurrence and severity of symptoms were confirmed through a survey during the test period. As a result, the results of the researcher's visual evaluation of the area where the test product was used and the results of the test subject's questionnaire were combined and evaluated.

As a result, as shown in Table 30 below, no special skin adverse reactions were confirmed as a result of the researcher's visual evaluation of the test area during the test period.

In addition, as shown in Table 31 below, as a result of a survey of test subjects, there were no special reports related to adverse skin reactions.

Example 7. Confirmation of skin absorption of cosmetic composition

Example 7.1. Selection of test subjects

Except for the condition (i) above, 21 adult women aged 20 to 59 who met the selection conditions and did not fall under the exclusion conditions were selected under the same conditions as in Example 6.1, as shown in Table 32 below.

The average age of the 21 test subjects was 48.762 years old, consisting of 11 people in their 40s and 10 people in their 50s. Additionally, the skin conditions of the 21 test subjects were 14 with dry skin, 4 with moderately dry skin, and 3 with neutral skin.

In addition, in the questionnaire regarding each test subject's experience with skin diseases, itchiness, stinging, erythema, side effects of cosmetics, side effects of medicines, photosensitivity, and atopic diseases, there were no test subjects who were eligible, and there were no test subjects who had experience with other items.

After explanation of the test, measurements were performed on the test subjects who agreed to participate in the test and signed the consent form, as shown in Table 33 below. After using the product, skin symptom evaluation was conducted. After the test was completed, a survey on effectiveness and preference was conducted.

Example 7.2. measurement method

For instrumental evaluation, the test subject rested for 30 minutes in a waiting room with constant temperature and humidity conditions of room temperature 20℃ to 24℃ and humidity 40 to 60% to adapt the skin surface temperature and humidity to the environment of the measurement space. During this time, water intake was limited. For objective measurement, measurements were made by one researcher, and the same area was measured for each measurement.

Example 7.3. Check skin absorption, absorption depth, and absorption speed

Skin absorption was measured using the 3D Raman Microscopy System Nanofinder ^® 30 (TOKYO INSTRUMENTS, Japan).

In particular, the 3D Raman system used in the test is a device designed to allow fine adjustment of spacing in the x, y, and z directions, and can move the z-axis focal length from the skin surface to the depth of the skin in micrometer units. Therefore, it is possible to obtain a 3D Raman spectrum image according to depth without damaging the test subject's skin, and using this, the skin absorption of cosmetics can be determined.

3D Raman scanning was performed by dividing two test/control areas of 3Х4 cm ² on the subject's left forearm and fixing the forearm to the equipment, and measurements were taken before and 30 minutes after using the product, respectively. The laser used for measurement was 785 nm, and the scanning step was fixed at 5 ㎛. Since it is set to capture 5 points in the x and y axis directions and 11 points in the z axis direction, the skin absorption of cosmetics can be known from the skin surface to a depth of 50 ㎛. Additionally, this can be converted into absorption depth (㎛) and absorption speed (㎛/h).

Skin absorption was evaluated in a total of 10 levels by dividing the z-axis into 10 equal parts at 5 ㎛ intervals on the 2D image. Therefore, the skin absorption depth can be converted by multiplying the change in skin absorption before and 30 minutes after using the product by 5 ㎛, and the skin absorption speed can be converted by dividing the absorption depth by the usage time of 0.5 hours.

As a result, as shown in Table 34, Figures 30 and 31, compared to before use, the skin absorption rate was statistically significant 30 minutes after using Composition 3 of the present invention (“Nudique Dia Spitera Ampoule” below). has been greatly improved.

In addition, Composition 3 has a remarkable 1.871-fold and 187% effect compared to the control group (“Nudique Spicule Ampoule” below) containing spicules without diamond coating, and a statistically significant difference is observed even after 30 minutes of use. These results mean that the cosmetic composition of the present invention with diamond coating has a significant skin absorption effect compared to the control group without diamond coating.

As a result of measuring the depth of skin absorption, as shown in Table 35 and Figures 32 and 33, it was confirmed that compared to before use, when composition 3 of the present invention was used, the skin absorption depth was 1.791 times and 179% deeper than the control group. . These results mean that the cosmetic composition of the present invention with diamond coating is absorbed into the skin more deeply than the control group without diamond coating, and the effect is significant.

As a result of measuring the skin absorption rate, as shown in Table 36 and Figures 34 and 35, it was confirmed that the absorption rate was 1.971 times and 179% faster when Composition 3 of the present invention was used compared to the control group compared to before use. did. These results mean that the diamond-coated cosmetic composition of the present invention is absorbed into the skin faster than the non-diamond-coated control group and has a significant effect.

Example 7.4. Validity Survey

After using the test product, the test subjects were asked to directly answer the survey data on the measurement items in 5 levels: very good (4), good (3), average (2), bad (1), and very bad (0). The researcher determined the efficacy of the test product by calculating the percentage of the number of test subjects for each answer.

As a result, as shown in Table 37 below, it was confirmed that the cosmetic composition of the present invention had a high effect of improving bodily sensation.

Example 7.5. Product preference survey evaluation

After using the test product, test subjects were asked to directly answer questionnaires about their feelings about using the product. The evaluation items were skin moisturization, skin smoothness, applicability, fragrance, and overall feeling of use on a 5-level scale: very good (4), good (3), average (2), bad (1), and very bad (0). .

As a result, as shown in Table 38 below, it was confirmed that the test subjects felt a high level of preference for the cosmetic composition of the present invention.

Example 7.6. Safety evaluation and adverse reaction evaluation

Safety evaluation and adverse reaction evaluation were conducted in the same manner as Example 6.22.

As a result, as a result of the researcher's visual evaluation of the test area during the test period, no special skin adverse reactions were confirmed. In addition, as a result of a survey of test subjects, there were no special reports related to adverse skin reactions, and no test subjects experienced adverse reactions during the test period.

Example 8. Confirmation of wrinkle improvement effect of cosmetic composition after sleep

Example 8.1. Selection of test subjects

Twenty-two test subjects were selected in the same conditions as in Example 6.1 above, except that there were wrinkles around the eyes, nasolabial folds, and neck wrinkles. All 22 people were female. As shown in Table 39 below, the average age was 48.818 years old, and there were 12 people in their 40s and 10 people in their 50s.

The skin conditions of the 22 test subjects were 10 with dry skin, 9 with medium dry skin, 1 with normal skin, and 2 with normal skin. There were no test subjects with skin diseases, itching, stinging, erythema, cosmetic side effects, pharmaceutical side effects, photosensitivity, or atopic disease.

The test subjects were instructed to use the test product after explaining how to use it. Measurements were conducted as shown in Table 40 below. After using the product, skin symptom evaluation was conducted. After the test was completed, a validity and preference survey was conducted.

Example 8.2. measurement method

For instrumental evaluation, the test subject rested for 30 minutes in a waiting room with constant temperature and humidity conditions of room temperature 20℃ to 24℃ and humidity 40 to 60% to adapt the skin surface temperature and humidity to the environment of the measurement space. During this time, water intake was limited. For objective measurement, measurements were made by one researcher, and the same area was measured for each measurement. All test subjects applied an appropriate amount of the cosmetic composition of Composition 3 (Nudique Dia Spitera Ampoule) to the skin once.

Example 8.3. Wrinkle relief effect when sleeping for 8 hours after applying the cosmetic composition

The test subject slept for 8 hours (12:00 pm to 8:00 am the next day) after application. Neck, nasolabial folds, and eye wrinkles were measured using a 3D skin imaging device, Antera 3D CS (Miravex Ltd, Ireland), on both sides of the subject's neck/nasolabial folds/eye area before and after using the test product and after 8 hours of sleep. For the analysis of wrinkles around the neck and around the eyes, the saved image was analyzed for the Indentation index (AU) value in Wrinkles (Medium) mode. At this time, as the Indentation index (AU) value decreases, it means that the wrinkles around the neck and around the eyes are improved. For wrinkle analysis, the average depth (mm) value of the saved image was analyzed in Wrinkles (Medium) mode. As the average depth (mm) value decreases, the nasolabial folds are improved.

As a result, as shown in Table 41 and Figure 36, neck wrinkles were improved by 9.767% immediately after use and 9.394% after sleep compared to before use of Composition 3 of the present invention. In addition, it was confirmed that the wrinkle improvement effect was more significant after 8 hours of sleep than the control group including spicules without diamond coating. These results mean that the cosmetic composition of the present invention with diamond coating has a significant improvement in neck wrinkles compared to the control group without diamond coating.

As a result, as shown in Table 42 and Figure 37, nasolabial folds were improved by 13.605% immediately after use and by 22.449% after sleep compared to before use of Composition 3 of the present invention. In addition, it was confirmed that the wrinkle improvement effect was more significant after 8 hours of sleep than the control group including spicules without diamond coating. These results mean that the cosmetic composition of the present invention coated with diamonds has a significant improvement in nasolabial folds compared to the control group that is not coated with diamonds.

As a result, as shown in Table 43 and Figure 38, wrinkles around the eyes were improved by 4.091% immediately after use and 9.794% after sleep compared to before use of Composition 3 of the present invention. In addition, it was confirmed that the wrinkle improvement effect was more significant after 8 hours of sleep than the control group including spicules without diamond coating. These results mean that the cosmetic composition of the present invention with diamond coating has a significant improvement in wrinkles around the eyes compared to the control group without diamond coating.

Example 8.4. Validity evaluation

After using the test product, participants were asked to directly answer the survey data in five levels: very good (4), good (3), average (2), bad (1), and very bad (0). The researcher determined the efficacy of the test product by calculating the percentage of the number of test subjects for each answer. The researcher determined the efficacy of the test product by calculating the percentage of the number of test subjects for each answer.

As a result, as shown in Table 44 below, it was confirmed that the cosmetic composition of the present invention had a high effect of improving physical sensation.

Example 8.5. Product preference survey evaluation

After using the test product, test subjects were asked to directly answer questionnaires about their feelings about using the product. The evaluation items are skin moisture, skin smoothness, applicability, absorption, fragrance, and overall feeling of use, with 5 levels: very good (4), good (3), average (2), bad (1), and very bad (0). It was evaluated as .

As a result, as shown in Table 45 below, it was confirmed that the test subjects felt a high level of preference for the cosmetic composition of the present invention.

Example 8.6. Safety evaluation and adverse reaction evaluation

Safety evaluation and adverse reaction evaluation were conducted in the same manner as Example 6.22 above.

As a result, as a result of the researcher's visual evaluation of the test area during the test period, no special skin adverse reactions were confirmed. In addition, as a result of a survey of test subjects, there were no special reports related to adverse skin reactions, and no test subjects experienced adverse reactions during the test period.

Claims (18)

Modified spicules coated with diamonds. According to paragraph 1,
A modified spicule containing an active ingredient inside the spicule. According to paragraph 2,
The active ingredient is collagen, a modified spicule. According to paragraph 1,
The modified spicule surface is coated with silica. According to paragraph 1,
The modified spicule surface is coated with an amino group. According to clause 5,
A modified spicule wherein the amino group coating is an aminosilane coating and/or a lysine coating. According to clause 6,
The aminosilane is 3-aminopropyltrimethoxysilane,
A modified spicule wherein the lysine is poly-L-lysine. According to paragraph 1,
The modified spicule surface is collagen-coated. A cosmetic composition comprising the modified spicule of claim 1 as an active ingredient. According to clause 9,
The cosmetic composition is for improving skin condition, improving wrinkles, improving elasticity, or moisturizing the skin. According to clause 10,
The cosmetic composition includes lotion (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, eye cream, hand cream, foundation, essence, nutritional essence. A cosmetic composition having the form of eye essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cream, body cleanser, suspension, gel, powder, paste, mask pack or sheet, or aerosol. (a) washing the spicule;
(b) enclosing the first active ingredient in the inner cavity of the cleaned spicule;
(c) coating the outside of the spicule containing the first active ingredient in the cavity with silica;
(d) reacting the silica-coated spicule with aminosilane and lysine to coat amino groups; and
(e) coating the amino group-coated silica with a second active ingredient and diamond; a method of producing a spicule coated with an ingredient effective for skin improvement and a diamond, comprising: According to clause 12,
A method for producing spicules, wherein the aminosilane in step (d) is 3-aminopropyltrimethoxysilane. According to clause 12,
A method for producing spicules, wherein the lysine in step (d) is poly-L-lysine. According to clause 12,
The mixing ratio of aminosilane and lysine in step (d) is 1:1 to 500:1. According to clause 12,
The diamond in step (e) is one or more selected from the group consisting of microdiamonds, nanodiamonds, and colloidal diamonds. According to clause 12,
The first active ingredient and the second active ingredient are each one or more selected from the group consisting of collagen, fish collagen, nanocollagen, low-molecular-weight collagen, vegetable collagen, collagen peptides, amino acids, tripeptides, tetrapeptides, and water-soluble vitamins,
A method for producing spicules, wherein the first active ingredient and the second active ingredient are the same or different ingredients. A spicule manufactured by the method of any one of claims 12 to 17.