KR102445188B1 - A functional micro-n-recovery cosmetic composition for effective skin regeneration and anti-aging comprising microneedle, low molecular weight peptide and natural extracts - Google Patents

Abstract

The present invention relates to a skin regeneration, skin whitening, wrinkle improvement, anti-aging and antioxidant cosmetic composition comprising fine needle powder, a low molecular weight peptide complex, and natural ingredients as active ingredients.

Description

A functional micro-and-recovery cosmetic composition containing fine needles, low-molecular peptides, and natural extracts for effective skin regeneration and anti-aging AND NATURAL EXTRACTS}

The present invention relates to an eco-friendly cosmetic composition comprising a natural fine needle powder and a low molecular weight peptide as active ingredients.

The skin is a membrane that covers the outside of the body and performs various physiological functions to protect the body from environmental factors such as various external stimuli, obstacles, and dryness, thereby protecting and regulating internal organs and other internal organs. do.

However, due to aging in the living body, the secretion of various hormones that control metabolism internally is reduced, and the function of immune cells and the activity of cells are reduced.

In addition, externally, the UV content in the sun's rays increases due to the destruction of the ozone layer, and the excessive physical and chemical stimulation and stress caused by the increase of free radicals and active harmful oxygen as the environmental pollution is further deepened is the normal function of the skin. It can damage the skin by promoting the production of spots, freckles, etc. and skin aging caused by deterioration, melanin deposition.

Our body performs a skin regeneration process on damaged skin. Skin regeneration is a tissue response to damage and is a process of tissue repair, such as chemotaxis, cell differentiation and replication, matrix protein synthesis, vascularization, and wound reconstitution. It is a complex biological process involving

Therefore, in order to prevent skin aging and damage caused by internal and external factors to maintain healthy and beautiful skin, physiologically active substances obtained from various animals, plants, microorganisms, etc. are added to cosmetics and used.

Efforts to maintain skin health by maintaining the intrinsic function of the skin and activating skin cells through various physiologically active substances as described above and promoting skin metabolism are continuing, and cosmetics and external preparations for skin are being developed accordingly.

However, these substances have limited usage due to safety issues such as irritation, erythema, and redness when applied to the skin, or have insignificant effects, making it difficult to actually expect skin function improvement effects.

Therefore, there is a demand for the development of a new composition for external application for skin that is safer and more effective than the existing composition for external application for skin.

Recently, in the cosmetic industry, a number of products using natural products have been developed to reduce skin irritation caused by chemicals. Natural ingredients have fewer side effects on the skin, and as consumers' response to cosmetics using natural ingredients increases, the value of development as a cosmetic raw material is increasing.

Therefore, the demand for cosmetics using natural ingredients with few side effects and high consumer response is increasing, and various studies on the development of multi-functional cosmetics with excellent biosafety and skin improvement effects are being actively conducted. have.

The present invention is to solve the problems of the prior art described above, and an object of the present invention is to provide a specialized functional cosmetic composition that maximizes the skin regeneration effect of the prior art by giving a physical and chemical double regeneration effect.

According to one aspect of the present invention, fine needle powder; And carnosine (carnosine) and chamomile extract; is provided a cosmetic composition comprising as an active ingredient.

In one embodiment, the cosmetic composition may further include palmitoyl tripeptide of SEQ ID NO: 1.

In one embodiment, the cosmetic composition may further include acetyl hexapeptide of SEQ ID NO: 2 (palmitoyl tripeptide).

In one embodiment, the cosmetic composition may further include at least one of a palmitoyl pentapeptide of SEQ ID NO: 3 and a palmitoyl tetrapeptide of SEQ ID NO: 4.

In one embodiment, the cosmetic composition may further include an extract of Centella asiatica or a callus extract of Resurrection plant.

In one embodiment, the cosmetic composition may be for anti-inflammatory or skin regeneration.

In one embodiment, the cosmetic composition may be for wrinkle improvement and skin elasticity improvement.

In one embodiment, the cosmetic composition may be for skin whitening or antioxidant.

The cosmetic composition according to one aspect of the present invention has a remarkably excellent skin improvement effect due to the synergistic effect of each active ingredient.

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

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, which may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like.

In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. All numerical limitations given throughout this specification will include all numerical ranges that are better within the broader numerical limits, as if the narrower numerical limitations were expressly written.

Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

One aspect of the present invention is a fine needle powder; And carnosine (carnosine) and chamomile extract; provides a cosmetic composition comprising as an active ingredient.

The cosmetic composition contains a natural peptide component as an active ingredient along with a natural extract, so that it has excellent biosafety and skin improvement activity as well as less skin irritation.

The "comprising as an active ingredient" may mean containing an effective amount that can exhibit a skin improvement effect, for example, wrinkle improvement, skin elasticity improvement, skin whitening, antioxidant effect, and the like.

The “Spicule” is a complex material composed of calcium and silicate in the form of various fine needles collected from sponges that usually live in freshwater in Europe and Russia, and can generally be obtained in the form of a powder. .

The microneedle has a fine needle-like structure and penetrates the skin layer to form a microchannel, so that the penetration effect of the applied cosmetic component can be significantly increased. The microneedle can promote the skin regeneration of collagen acting on artificial wound healing and increase the bioactivity by increasing the skin tissue temperature.

The fine needle is not particularly limited as long as it is a material that is harmless to the human body and is a powdery material that can be dissolved or discharged from the body by stimulating the skin when in contact with the skin to form fine pores.

Spongilla lacustris L. , Spongilla fragilis Leidy and Ephydatia fluviatilis ).

The sponges are primitive marine organisms that do not have muscles, nerves, or organs, and there are more than 5000 species. Bone fragments are needle-shaped skeletal tissues that exist in the body of invertebrates and are mainly composed of silicate and calcium carbonate.

The sponge may include uniaxial, triaxial, tetraaxial, or multiaxial bone fragments containing calcium carbonate or silicic acid as a main component.

The sponges mainly inhabit the sea in the form of sponges of various colors such as orange or turquoise, but freshwater sponges also exist.

The spongy animal is an organism in the initial stage of tissue in a state in which cells are aggregated, and the body is composed of sponge fibers, spicules forming the body wall, and a cell group bound to the skeletal fragments. The main component of the bone fragment is calcareous, silicic acid or silica, and the size is about 100 μm, so it can be observed under a microscope.

As an effective means of skin regeneration, the bone fragments can be used to correct various etiologies of hyperpigmentation, fine wrinkles, sun damage, and visual facial defects such as superficial scars, comedones, and enlarged pores.

In addition, the bone fragments are being utilized as natural alternative approaches to microdermabrasion and dermabrasion, chemical dermabrasion, CO ₂ laser dermabrasion, and erbium laser dermabrasion, which are physical procedures to improve the skin condition.

When the bone fragments are rubbed against the skin layer, they are physically separated to reduce keratinocyte aggregation, and the stratum corneum, sebum plug, and keratinocytes are detached to form micro passages, thereby maximizing the penetration effect of cosmetic ingredients.

The carnosine is a dipeptide of β-alanyl-L-histidine composed of β-alanin and histidine, and is found in a fairly large amount in long-lived tissues, especially in the spine. It is abundantly present in the non-protein fraction of animal muscle cells, and the amount ranges from about 1 to 20 mM.

As an antioxidant that protects cells from oxidative stress, canosine is known as a free radical scavenger having an excellent effect in removing reactive oxygen species.

The protective action of canosine is achieved by inhibiting the transformation of biopolymers in an oxidative stress environment through proton buffering, heavy metal chelating, and removal of free radicals and active sugar molecules and maintaining their intrinsic function. The present inventors have identified that the canosine can exhibit an excellent synergistic effect by acting with the fine needles and natural extracts.

The "chamomile ( Matricaria chamomilia )" is a perennial herbal plant belonging to the Asteraceae, which is native to India and Europe, and includes yellow flowers of diamonds chamomile, white flowers of Roman chamomile, German chamomile, bode gold chamomile, and the like.

The chamomile is widely used as a bathing agent in Europe because of its great skin sterilization effect and whole body beauty effect, is effective for poor circulation in women, and is used as an antiseptic, anthelmintic and spasm relief. In addition, the chamomile is also known to have effects such as prevention of colds due to sweating, relieving insomnia, and sedation as a gastric medicament.

The "extract" is an active ingredient contained in the extraction raw material by contacting the solvent and the extraction raw material under specific conditions, and as long as it is a substance obtained by separating the ingredients contained in the raw material from the natural product, it may be included regardless of the extraction method or the type of ingredient .

For example, extracting a component soluble in a solvent from a natural product using water or an organic solvent, a specific component of a natural product, for example, extracting only a specific component such as oil, etc. may be included. The extract can be obtained by drying the natural raw material and then pulverizing it, or by various conventionally known extraction methods such as hot water extraction and ethanol extraction.

The extract is selected from the group consisting of water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, hexane and 1,3-butylene glycol It may be extracted with one or more solvents, specifically, 60 to 80% (v/v) of ethanol.

The extraction ratio of the active ingredients contained in the raw material may be different depending on the polarity of the solvent, and since the ethanol has excellent selectivity in extracting the physiologically active substances of natural raw materials, the optimal skin improvement effect may be realized by the ethanol extraction. can

Since the water and ethanol have different polarities and the active ingredients extracted according to each polarity may vary, the concentration of the ethanol may be appropriately controlled so that an optimal skin improvement effect can be realized.

At this time, if the concentration of the ethanol is less than 60%, the active ingredient showing the skin improvement effect may not be sufficiently extracted, and if it exceeds 80%, an appropriate yield may not be realized.

The extract is extracted and filtered by conventional methods such as reflux circulation extraction, pressure extraction, ultrasonic extraction, etc. for 24 to 240 hours with a solvent in a volume equivalent to 10 to 20 times the weight of the raw material by washing the raw material for extraction with water, drying and pulverizing it can be manufactured. In addition, the extract may be obtained in a powder state by an additional process such as distillation under reduced pressure or freeze-drying.

The extract may also include an extract that has undergone a conventional purification process. For example, the extract is separated using an ultrafiltration membrane having a constant molecular weight cut-off value, and various purification methods additionally performed such as separation by various chromatography (those prepared for separation according to size, charge, hydrophobicity or affinity). It may include a fraction obtained through

The cosmetic composition may further include a peptide (peptide) for skin improvement.

The peptide refers to a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds). For the purpose of the present invention, the peptide may refer to a peptide or a fragment thereof that exhibits wrinkle improvement and skin elasticity improvement effects.

A general rule for naming such peptides may be based on a three-letter or one-letter amino acid code, unless specifically indicated exception. For example, the central part of the amino acid structure is indicated by a three-letter code (eg, Ala, Lys), and a D-conformation (eg, D-Ala, D-Lys) is specified by writing "D-" in front of the three-letter code. If not indicated, it can be assumed to have an L-stereoscopic shape. The amino acid residues constituting the peptide may be natural or non-natural amino acid residues.

The present inventors have identified that the peptide can effectively inhibit skin damage and skin aging that may be caused by various factors by interacting with other components, and various uses have been derived through this.

The peptide can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (Merrifield, J. Amer. Chem. 1963, Soc. 85:2149-54; Stewart, et al. Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. 1984, Co.: Rockford, 111).

The cosmetic composition may include palmitoyl tripeptide of SEQ ID NO: 1.

The "palmitoyl tripeptide" is a reaction product of palmitic acid and tripeptide and may consist of an amino acid sequence represented by Pal-Gly-His-Lys (SEQ ID NO: 1), and collagen It is a peptide that mimics a fragment of structure 1.

The palmitoyl tripeptide may activate extracellular matrix (ECM) cells and regenerate collagen, restore skin intercellular lipids to reduce wrinkles, and alleviate aging symptoms without hypersensitivity symptoms.

The cosmetic composition may include acetyl hexapeptide of SEQ ID NO: 2 (palmitoyl tripeptide).

The "acetyl hexapeptide (Acetyl Hexapeptide)" may be composed of the amino acid sequence represented by Ac-Glu-Glu-Met-Gln-Arg-Arg-NH ₂ (SEQ ID NO: 2), and can be obtained through various methods. and, preferably, it can be obtained by solid phase peptide synthesis.

The common name of the acetyl hexapeptide is argirenline, and it can be used as a cosmetic composition for wrinkle improvement. For example, the acetyl hexapeptide may prevent the generation of wrinkles by inhibiting a reaction such as movement or contraction of a facial muscle.

The cosmetic composition may further include at least one of a palmitoyl pentapeptide of SEQ ID NO: 3 and a palmitoyl tetrapeptide of SEQ ID NO: 4.

The "palmitoyl pentapeptide" is a reaction product of palmitic acid and a pentapeptide, and is composed of an amino acid sequence represented by Pal-Lys-Thr-Thr-Lys-Ser (SEQ ID NO: 3). The "palmitoyl tetrapeptide" may be composed of an amino acid sequence represented by Pal-Gly-Gln-Pro-Arg (SEQ ID NO: 4).

The cosmetic composition may further include an extract of Centella asiatica or an extract of Resurrection plant.

The " Centella Asiatica " is a perennial plant commonly found in the mountains or fields of the southern islands of Korea. The main stem extends sideways, and there are two degenerated scaly leaves near the root node.

The main components of Centella asiatica are beta-carotene, beta-sitosterol, campesterol, kaempferol, saponin, and the like. It is known to be effective against fever and fever.

The resurrection plant is also called the rose of Jericho because it grows in the 'Jericho' valley of the Judean wilderness in the Bible, and the trihalose component helps the resurrection plant to live in extreme environments.

Since the trihalose contains a lot of hydroxy (OH) groups in the molecule, it is known that it has an excellent effect in strengthening skin moisturizing power because it can attract a lot of water.

The resurrection plant extract may be an resurrection plant callus extract using the resurrection plant callus as an extraction raw material.

The "resurrection plant callus" may be any part derived from any part of the resurrection plant, but in one embodiment, it may be a callus induced by cutting cotyledons from the germinated seedlings of the resurrection plant seeds, and any tissue of the resurrection plant Callus can be formed by cutting it and placing it in a medium with the content of auxin and cytokinin adjusted.

The cosmetic composition may be for anti-inflammatory or skin regeneration.

The “anti-inflammatory” refers to the action of inhibiting inflammation, and the regulation of the inflammatory response is known to be very complex, which reduces the enhancement and damage of the repair system in vivo.

If the inflammatory response continues due to repeated tissue damage or regeneration, ROS and RNS are overproduced in inflammation-related cells, and permanent gene modification can be caused. That is, ROS and RNS are deeply related to the inflammatory response that regulates the actions of various cells in vivo.

The cosmetic composition may exhibit excellent anti-inflammatory activity by regulating the expression of inflammatory mediators generated due to the activation of NF-kB.

The “skin regeneration” refers to a process of restoring skin tissue from damage caused by external and internal causes of the skin.

The damage caused by the external cause may include ultraviolet rays, external pollutants, wounds, trauma, and the like, and the damage caused by the internal cause may include stress.

The cosmetic composition may restore the skin barrier, promote skin regeneration, and increase moisture to protect the skin from UV rays or pollution, increase immunity, and inhibit skin aging.

The cosmetic composition may be for wrinkle improvement and skin elasticity improvement.

The "improvement of skin elasticity" refers to a phenomenon in which elastic fibers composed of elastin exist together with collagen, and skin elasticity is maintained or improved in a state in which the elastin and collagen are sufficiently present.

In addition, the skin elasticity improvement effect and the skin wrinkle improvement effect may be accompanied at the same time. The "wrinkle improvement" refers to a phenomenon that suppresses or inhibits the generation of wrinkles on the skin, or alleviates the already generated wrinkles.

The cosmetic composition may be for skin whitening or antioxidant.

The "skin whitening" suppresses the production of various biomolecules such as melanin, oxidation-reduction hemoglobin, carotene, and melanoids that affect skin color, thereby alleviating skin pigmentation phenomena such as blemishes, freckles, and melasma. It refers to the effect of improving skin brightness and uniformity by alleviating redness.

The “antioxidant” refers to the action of inhibiting oxidation, and the human body is in a balance between the oxidation promoting substance and the oxidation inhibitory substance. , oxidative stress is induced in the living body, which can cause cell damage and pathological diseases.

Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is chemically unstable and highly reactive, so it can easily react with various biomaterials such as DNA, proteins, lipids and carbohydrates, It attacks and causes irreversible damage to cells and tissues or causes mutations, cytotoxicity and cancer, and is also a direct cause of aging.

The cosmetic composition can prevent aging and maintain health by obtaining an antioxidant effect by removing or reducing the reactive oxygen species.

The cosmetic composition includes a softening lotion, a nourishing lotion, a moisture cream, a nourishing cream, a massage cream, a nourishing lotion, an essence, an ampoule, a gel, an eye cream, an oil, a foundation, a cleansing cream, a cleansing foam, a cleansing water, a shampoo, a conditioner, a pack, It may be formulated with one or more selected from the group consisting of sprays and powders.

The cosmetic composition may be formulated by a conventional method. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, the International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association) , Inc., Washington, 1995) may be referred to.

Specifically, the cosmetic composition can be prepared in general emulsified formulations and solubilized formulations. For example, lotion, such as a softening lotion or a nourishing lotion; emulsions such as facial lotions and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Or it may be formulated as a cleaning agent such as a cleansing foam, soap, body wash, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the cosmetic composition, in addition to the essential components in each formulation, other components may be appropriately blended within the range that does not impair the purpose according to the present invention, depending on the type of formulation or purpose of use.

The cosmetic composition may include a conventionally acceptable carrier, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately mixed, but is limited thereto not.

The acceptable carrier may vary depending on the formulation. For example, when formulated into an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or their Mixtures can be used

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or mixtures thereof may be used as a carrier component, and in the case of spray, chlorofluorohydro It may further contain a propellant such as carbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-butylglycol oil may be used.

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

The cosmetic composition is a fatty substance, organic solvent, solubilizer, thickening agent, gelling agent, softening agent, antioxidant, suspending agent, stabilizing agent, foaming agent, and fragrance commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, commonly used in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as any other ingredients.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

Through the following examples, the present invention will be described in more detail, but it is obvious that the present invention is not limited by the following examples.

Preparation Example 1: Preparation of fine needle powder

800 mL of 0.5N-sodium hydroxide solution was added to 200 g of a natural sponge obtained from dried sponge ( Songilla lacustris ), and mixed and pulverized by an ultrasonic process.

After stirring, impurities were removed by centrifugation, and 700 mL of purified water was added to remove soluble impurities and sodium hydroxide solution.

A small amount of sodium hydroxide was chemically removed with 800 mL of a 2%-L-ascorbic acid solution, and the over-injected L-ascorbic acid solution was removed and dried to obtain a fine needle powder from which purified water was removed.

Preparation Example 2: Preparation of natural ingredients

After washing chamomile, centella asiatica, and resurrection plant callus with water, it was dried completely at room temperature and pulverized to obtain 100 g of each pulverized product.

For each pulverized product and raw material, 10 times the volume of an aqueous ethanol solution (70% w/w) was used as a solvent and immersion extraction was performed at a temperature of 50° C. for 12 hours.

After the extract was filtered through a 250 mesh and 0.5 μm filter, the remaining raw materials were extracted three times in the same manner, concentrated under reduced pressure at 30° C., and freeze-dried to obtain a solid content of the natural extract.

Examples and Comparative Examples

Examples and Comparative Examples were set by mixing samples as shown in Tables 1 and 2 below to verify the skin improvement effect of the obtained extract.

Each peptide was obtained from the market and used.

[Content (parts by weight)] division Example One 2 3 4 5 6 7 8 9 fine needle powder 300 300 300 300 300 300 300 300 300 carnosine 150 100 75 75 60 50 50 50 37.5 chamomile extract 150 100 75 75 60 50 50 50 37.5 Palmitoyl tripeptide (SEQ ID NO: 1) - 100 75 75 60 50 50 50 37.5 Acetyl Hexapeptide (SEQ ID NO: 2) - - 75 - 60 50 50 50 37.5 Palmitoyl tripeptide (SEQ ID NO: 3) - - - 75 60 50 50 50 37.5 Palmitoyl tetrapeptide (SEQ ID NO: 4) - - - - - 50 - - 37.5 Centella asiatica extract - - - - - - 50 - 37.5 Resurrection Plant Callus Extract - - - - - - - 50 37.5

[Content (parts by weight)] division comparative example One 2 3 4 5 6 7 8 9 fine needle powder 300 - - - 300 - 300 - - carnosine - 300 - - - 150 150 100 - chamomile extract 300 - 300 150 - 100 - Palmitoyl tripeptide (SEQ ID NO: 1) - - - - - 150 100 - Acetyl Hexapeptide (SEQ ID NO: 2) - - - - - - - - 60 Palmitoyl tripeptide (SEQ ID NO: 3) - - - - - - - - 60 Palmitoyl tetrapeptide (SEQ ID NO: 4) - - - - - - - - 60 Centella asiatica extract - - - - - - - - 60 Resurrection Plant Callus Extract - - - - - - - - 60

Experimental Example 1: Skin safety test

In order to verify the skin safety of Examples and Comparative Examples, MTT assay experiments were performed on fibroblasts (HDF), melanocytes (B16F10), and keratinocytes (HaCaT).

After each of the samples of Examples and Comparative Examples were suspended in purified water at concentrations of 0, 1, 5, 10, 20, 50 and 100 mg/L, cell viability was measured.

Cytotoxicity was performed by modifying Mosmann's method for measuring cell viability using MTT {3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide} reagent.

Fibroblasts (HDF), melanocytes (B16F10), and keratinocytes (HaCaT) were each dispensed in a 96-well plate at a concentration of 1 × 10 ⁴ cells/well and cultured at 37° C., 5% CO ₂ for 48 hours. did.

After removing the culture medium and culturing the sample in a medium treated for each concentration for 48 hours, the medium was removed and washed repeatedly with PBS (Phosphate buffered saline).

MTT was dissolved in PBS at 5mg/mL, 50L was added, and incubated at 37°C, 5% CO ₂ for 48 hours. 100 L of DMSO (dimethyl sulfoxide) was added per well, and the absorbance was measured at 540 nm after stirring for 10 minutes.

As a result of the measurement, cytotoxicity was not confirmed at all concentrations of each sample. The above results suggest that the sample is not only harmless to the human body, but also has excellent human safety.

Experimental Example 2: Evaluation of free radical scavenging activity

The extracts of Examples and Comparative Examples were suspended in purified water to evaluate free radical scavenging activity.

The DPPH measurement method is a method of measuring the absorbance at 540 nm to determine the extent to which an inhibitor is decolorized by scavenging a stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl radical).

Vitamin C was used as a control, and the same experiment was repeated 3 times for the accuracy of the experiment, and the results are shown in Table 3 below.

[Concentration that scavenges 50% of radical DPPH] division SC ₅₀ (μg/mL) Example 1 130.1 Example 2 119.4 Example 3 116.8 Example 4 116.4 Example 5 115.7 Example 6 113.1 Example 7 114.4 Example 8 112.9 Example 9 111.0 Comparative Example 1 220.9 Comparative Example 2 193.2 Comparative Example 3 181.5 Comparative Example 4 180.4 Comparative Example 5 177.5 Comparative Example 6 170.6 Comparative Example 7 173.7 Comparative Example 8 168.6 Comparative Example 9 170.5 Control (Vitamin C) 125.3

The lower the SC ₅₀ value, the higher the scavenging activity of DPPH free radicals.

Referring to Table 3, the sample of Example was significantly superior to the effect of improving skin elasticity compared to Comparative Example.

In particular, Example 1 containing fine needle powder, carnosine and chamomile extract at the same time had significantly superior free radical scavenging activity compared to Comparative Examples 5 to 7 lacking a portion, and the result was a synergistic effect according to the combination of each component. suggests

Experimental Example 3: Evaluation of anti-inflammatory activity

The anti-inflammatory activity of Examples and Comparative Examples was evaluated by analyzing the NO production inhibitory ability of EAME in the rat macrophage RAW 264.7 cell induced by lipopolysaccharide (LPS) stimulation.

For the experiment, Raw 264.7 cells, a murine macrophage cell line, were distributed from the Korean cell line bank.

Using DMEM medium containing 100 units/mL penicillin-streptomycin and 10% fetal bovine serum (FBS), it was cultured in an incubator at 37° C., 5% CO ₂ , and subculture was performed once every 2-3 days.

Raw 264.7 cells (3 x 10 ⁵ cells/mL) were cultured 18 hours before, and samples of Examples and Comparative Examples (concentration 10%) and LPS (1 μg/mL) were co-treated and cultured for 24 hours.

The generated NO was measured in the form of NO ₂ ⁻ present in the cell culture medium using Griess reagent.

The negative control group was not treated with LPS, and the positive control group was treated with only LPS to measure the amount of NO generated.

100 μL of cell culture supernatant and Griess reagent (1% (w/v) sulfanilamide, 0.1% (w/v) naphtylehtylenediamine in 2.5% (v/v) phosphoric acid) were mixed in equal amounts and reacted in the dark at room temperature for 10 minutes, followed by ELISA Absorbance (540 nm) was measured using a reader.

The standard concentration curve was calculated by serial dilution of sodium nitrite (NaNO ₂ ) (10-100 μM). The measurement results are shown in Table 4 below.

division NO production rate (%) Example 1 53.6 Example 2 52.4 Example 3 51.7 Example 4 51.3 Example 5 50.3 Example 6 48.1 Example 7 49.2 Example 8 47.2 Example 9 44.4 Comparative Example 1 89.8 Comparative Example 2 83.4 Comparative Example 3 79.9 Comparative Example 4 75.8 Comparative Example 5 74.5 Comparative Example 6 74.0 Comparative Example 7 72.7 Comparative Example 8 75.2 Comparative Example 9 76.0 control 31.0 negative control (no treatment) 100.0

Referring to Table 4, the samples of Examples 1 to 8 had excellent NO (nitric oxide) production inhibitory activity compared to Comparative Examples 1 to 9.

In particular, Example 1 containing fine needle powder, carnosine and chamomile extract at the same time had significantly superior anti-inflammatory activity compared to Comparative Examples 5 to 7 lacking some, and the results suggest a synergistic effect according to the combination of each component. do.

Experimental Example 4: Evaluation of melanin production inhibitory activity

In order to evaluate the melanogenesis inhibitory effect of Examples and Comparative Examples, melanocytes B16F10 were cultured.

B16F10 was dispensed into each well of a 6-well plate to make 8 x 10 ⁴ cells, and then incubated for 18 hours in a cell incubator under 5% CO ₂ , 37°C conditions.

After 18 hours, the medium of the B16F10 cells was removed, and the samples of Examples and Comparative Examples were treated at the same concentration and cultured for 2 days in a cell incubator at 5% CO ₂ , 37° C. conditions.

After 2 days, cells were recovered from the plate using a scraper and centrifuged for 1 minute to obtain cells in the form of pellets.

1N NaOH was added to the obtained cells and reacted at 95° C. for 10 minutes to dissolve melanin.

After 10 minutes, the amount of melanin production was measured at a wavelength of 475 nm using a microplate reader, and the amount of protein was measured at a wavelength of 595 nm using a BCA assay.

Each experimental group was expressed numerically as “absolute amount of melanin / absolute amount of protein”, and the experimental group and control group were quantified relative to each other.

α-MSH was used as a melanogenesis inducer, and arbutin was used as a positive control.

division Melanin Relative Value (%) Example 1 53.6 Example 2 52.4 Example 3 51.1 Example 4 51.5 Example 5 50.7 Example 6 49.7 Example 7 48.2 Example 8 47.5 Example 9 46.3 Comparative Example 1 89.8 Comparative Example 2 81.3 Comparative Example 3 76.8 Comparative Example 4 75.8 Comparative Example 5 74.5 Comparative Example 6 74.0 Comparative Example 7 75.8 Comparative Example 8 75.2 Comparative Example 9 74.9 positive control 43.3 control 100.0

Referring to Table 5, the samples of Examples 1 to 8 were excellent in melanin production inhibitory activity compared to Comparative Examples 1 to 9.

In particular, Example 1 containing fine needle powder, carnosine and chamomile extract at the same time had significantly superior melanin production inhibitory activity compared to Comparative Examples 5 to 7 lacking some, and the result was synergistic according to the combination of each component of the present application suggest an effect.

Experimental Example 5: Tyrosinase (tyrosinase) inhibitory activity evaluation

Tyrosinase inhibitory activity of the Examples and Comparative Examples was evaluated.

Inhibitory activity was measured by treatment with the same concentration, and L-DOPA was used as a substrate.

Dissolve mushroom tyrosinase in 68mM sodium phosphate buffer (pH 6.8) to 125U/ml, and store at -20℃ for use in the experiment.

In a 96-well plate, 40 μM of diluted sample and 120 μL of 8 mM L-DOPA were added.

After adding 40 μL of mushroom tyrosinase and reacting for 20 minutes, absorbance was measured at 490 nm with an ELISA reader.

As a control, kojic acid was used. Tyrosinase inhibitory activity was measured by measuring the absorbance ratio of the sample addition group compared to the sample non-addition group.

division Tyrosinase activity relative value (%) Example 1 47.7 Example 2 45.7 Example 3 44.8 Example 4 45.3 Example 5 44.5 Example 6 43.1 Example 7 41.8 Example 8 41.3 Example 9 39.6 Comparative Example 1 92.2 Comparative Example 2 81.5 Comparative Example 3 76.7 Comparative Example 4 77.6 Comparative Example 5 76.1 Comparative Example 6 73.4 Comparative Example 7 78.1 Comparative Example 8 76.8 Comparative Example 9 74.5 positive control 39.4 control 100.0

Referring to Table 6, the samples of Examples 1 to 8 exhibited superior tyrosinase inhibitory activity compared to Comparative Examples 1 to 9. In particular, Example 1 containing fine needle powder, canosine, and chamomile extract simultaneously showed some The tyrosinase inhibitory activity was significantly superior to those of Comparative Examples 5 to 7 lacking in , and the results suggest the excellent whitening activity of the present invention.

Experimental Example 6: Elastase inhibitory effect evaluation

The wrinkle improvement effect of the samples of Examples and Comparative Examples was evaluated through the inhibitory activity of elastase. The samples of Examples and Comparative Examples were suspended in purified water and elastase inhibitory activity was measured as follows.

1.6 mM elastase substrate (N-succinyl-(Ala)3-p-nitroanilide; SANA) and 0.2 mM Tris-HCl buffer (pH 8.0) were prepared. 0.165 mL of the Tris-HCl buffer, 0.005 mL of SANA, and 0.01 mL of elastase were dispensed into 96 well-plates.

After putting the samples of Examples and Comparative Examples together with a positive control and reacting at 25° C. for 15 minutes, absorbance was measured at 405 nm.

The percentage (%) of elastase inhibitory activity was calculated as the ratio of the difference between the absorbance to which the sample was added to the absorbance to which the sample was not added.

As a positive control, oleanolic acid was used, and the concentration (IC ₅₀ ) that inhibited elastase by 50% of the samples of Examples and Comparative Examples was measured and shown in Table 7.

[Elastase 50% inhibitory concentration] division IC ₅₀ (μg/mL) Example 1 160.2 Example 2 152.2 Example 3 148.6 Example 4 148.9 Example 5 145.0 Example 6 138.2 Example 7 135.3 Example 8 134.8 Example 9 125.8 Comparative Example 1 - Comparative Example 2 332.4 Comparative Example 3 285.4 Comparative Example 4 338.5 Comparative Example 5 284.1 Comparative Example 6 295.2 Comparative Example 7 277.7 Comparative Example 8 281.4 Comparative Example 9 293.0 positive control 96.8

The lower the IC ₅₀ value, the higher the elastase activity inhibition rate.

Elastase is an enzyme that breaks down elastin, an important substrate for maintaining skin elasticity in the dermis. In addition, elastase is a non-specific hydrolase capable of degrading collagen (collagen), which is another important matrix protein.

Therefore, the high elastase activity inhibition rate means that elastin and collagen, which are important for maintaining skin elasticity, are maintained for a long time, suggesting that the skin wrinkle improvement effect is excellent.

Referring to Table 7, the samples of Examples more effectively inhibited elastase activity compared to Comparative Examples.

Experimental Example 7: Evaluation of wrinkle improvement effect

The skin wrinkle improvement effect was evaluated through the following experiment.

Adult women over 30 years of age were divided into 18 groups, and the test subject was thoroughly absorbed by applying the cream-formed cosmetic to the face after cleansing every morning and evening for 4 weeks.

ANTERA 3D (Miravex, Ireland) was applied to evaluate wrinkle improvement, and device measurements were made before and after use of the test substance for 3 weeks.

The same test person measured the left corner of the eye of all subjects, and for reproducibility of the measurement, the same part was measured by overlapping the image measured before the test substance was used.

The captured images were matched using ANTERA pro software, which is a dedicated software for ANTERA 3D, and then the matched measurement area was used for analysis.

Wrinkles small values representing skin wrinkles were analyzed using the indentation index, which is a measurement variable.

Table 8 shows the amount of change in Wrinkles small value after 4 weeks of the test subject according to the use of cosmetics formulated with samples of Examples and Comparative Examples. The greater the wrinkle improvement effect, the greater the change in the Wrinkles small value.

division Wrinkles small average Example 1 1.20 Example 2 1.24 Example 3 1.27 Example 4 1.26 Example 5 1.29 Example 6 1.35 Example 7 1.34 Example 8 1.34 Example 8 1.44 Comparative Example 1 0.45 Comparative Example 2 0.64 Comparative Example 3 0.81 Comparative Example 4 0.79 Comparative Example 5 0.71 Comparative Example 6 0.74 Comparative Example 7 0.79 Comparative Example 8 0.75 Comparative Example 9 0.73

Referring to Table 8, the cosmetic formulated with the sample of Example was significantly superior to the skin wrinkle improvement effect compared to Comparative Example.

Experimental Example 8: Evaluation of skin elasticity improvement effect

The skin elasticity improvement effect of the cosmetic compositions formulated with the samples of Examples and Comparative Examples was evaluated.

Each of the samples was formulated as a cream of the same composition, and only the samples of Examples and Comparative Examples were different.

The creams of Examples and Comparative Examples prepared by dividing into groups of healthy women (average age of 37 years) over 20 years of age at a temperature of 24 to 26° C. and a humidity of 75% were applied to each group.

The cream formulated for 12 weeks was applied twice a day (morning and evening) around the eyes, and skin elasticity was measured using a skin elasticity meter (Cutometer MPA580, Conrage + Khazaka, Germany).

The test results were described as R8 (R8 (12 weeks)-R8 (0 weeks)) values of the cutometer MPA580, and the R8 values represent the properties of skin viscoelasticity, and the results are shown in Table 9 below.

division Skin elasticity improvement effect Example 1 0.74 Example 2 0.78 Example 3 0.80 Example 4 0.81 Example 5 0.84 Example 6 0.86 Example 7 0.86 Example 8 0.89 Example 9 0.93 Comparative Example 1 0.28 Comparative Example 2 0.42 Comparative Example 3 0.41 Comparative Example 4 0.40 Comparative Example 5 0.43 Comparative Example 6 0.46 Comparative Example 7 0.44 Comparative Example 8 0.50 Comparative Example 9 0.39 Positive control (vitamin C) 0.91 negative control (no treatment) 0.31

Referring to Table 9, the sample of Example was significantly superior to the skin elasticity improvement effect compared to Comparative Example.

In particular, Example 1 containing fine needle powder, carnosine and chamomile extract at the same time was significantly superior to Comparative Examples 5 to 7 lacking some, and the results suggest a synergistic effect according to the combination of each component.

Experimental Example 9: Evaluation of wound healing effect by collagen biosynthesis

Male Sprague-Dawley rats (weight around 200 g) were anesthetized with sodium pentobarbital.

After shaving the rat's back hair and sterilizing the skin with tincture of iodine, two symmetrical circular excision windows were made on the back skin using a circular (inner diameter 1 cm) leather punch.

From the day of the wound onset, hydrocortisone was administered subcutaneously daily at a dose of 50 mg/kg once a day to delay wound healing.

Only the samples of Examples and Comparative Examples were formulated into creams of the same composition, and the wound healing effect was evaluated.

300 mg of the cream was applied once a day to one place of the wound from the day of the occurrence of the wound.

After 8 days, the area of the wound was measured, compared with the untreated control group, and the wound healing accelerating rate was calculated by substituting the measured area of the wound into the following equation.

[Equation]

Healing acceleration rate (%) = 100- (test group wound area / control group wound area x 100)

division Wound healing promotion rate (%) Example 1 59.3 Example 2 61.8 Example 3 63.4 Example 4 64.2 Example 5 66.7 Example 6 68.3 Example 7 68.3 Example 8 69.8 Example 9 74.0 Comparative Example 1 18.5 Comparative Example 2 27.7 Comparative Example 3 27.2 Comparative Example 4 26.6 Comparative Example 5 28.3 Comparative Example 6 30.6 Comparative Example 7 29.5 Comparative Example 8 33.5 Comparative Example 9 26.0

Referring to Table 10, the sample of Example reduced the wound area very effectively compared to Comparative Example (P<0.01).

In particular, Example 1 containing fine needle powder, carnosine and chamomile extract at the same time was significantly superior to Comparative Examples 5 to 7 lacking some, and the results showed that the wound healing effect was improved by synergy according to the combination of each component. suggest that it has been

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

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

SEQUENCE LISTING <110> KIM, HYEYONG <120> A FUNCTIONAL MICRO-N-RECOVERY COSMETIC COMPOSITION FOR EFFECTIVE SKIN REGENERATION AND ANTI-AGING COMPRISING MICRONEEDLE, LOW MOLECULAR WEIGHT PEPTIDE AND NATURAL EXTRACTS <130> P20-0066 <160> 4 <170> PatentIn version 3.2 <210> 1 <211> 3 <212> PRT <213> ARTIFICIAL <220> <223> ARTIFICIAL <400> 1 Gly His Lys One <210> 2 <211> 6 <212> PRT <213> ARTIFICIAL <220> <223> SEQ ID 3 <400> 2 Glu Glu Met Gln Arg Arg 1 5 <210> 3 <211> 5 <212> PRT <213> ARTIFICIAL <220> <223> ARTIFICIAL <400> 3 Lys Thr Thr Lys Ser 1 5 <210> 4 <211> 4 <212> PRT <213> ARTIFICIAL <220> <223> ARTIFICIAL <400> 4 Gly Gln Pro Arg One

Claims (8)

fine needle powder;
Palmitoyl tripeptide of SEQ ID NO: 1, acetyl hexapeptide of SEQ ID NO: 2, palmitoyl pentapeptide of SEQ ID NO: 3, and palmitoyl tetrapeptide of SEQ ID NO: 4 a complex peptide consisting of; and
Carnosine (carnosine), chamomile extract, Centella asiatica extract, and Resurrection plant callus extract; Anti-inflammatory and antioxidant cosmetic composition comprising as an active ingredient. delete delete delete delete According to claim 1,
A cosmetic composition further comprising the use of skin regeneration. According to claim 1,
A cosmetic composition further comprising the purpose of improving wrinkles and improving skin elasticity. According to claim 1,
A cosmetic composition further comprising a skin whitening use.