Cosmetic composition containing low molecular weight anionic peptides separated from pomelo seeds

Abstract

The invention relates to a cosmetic composition containing low molecular weight anionic peptides separated from grapefruit seeds. More specifically, the invention relates to a preparation method for separating low-molecular-weight peptides from grapefruit seeds and then processing the low-molecular-weight peptides into low-molecular-weight anionic peptides, and a cosmetic composition containing the low-molecular-weight peptides as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a cosmetic composition containing a low molecular weight anionic peptide isolated from grapefruit seeds, and more particularly to a preparation method of a low molecular weight anionic peptide after separation from grapefruit seeds and processing the low molecular weight anionic peptide, and a cosmetic composition containing the low molecular weight anionic peptide as an effective ingredient. Background Art

[0002] It is known that the numerous pollutants in the atmosphere generated by the combustion of automobiles, boilers, power generation facilities, etc. and the fine dust emitted thereby not only cause various diseases, but also cause inflammation, induce allergies and accelerate skin aging by damaging the skin barrier. The above-mentioned fine dust generally refers to all pollutants of very small size. The size of fine dust particles is less than 10 μm in diameter. Not only is it difficult to confirm with the naked eye, but also it is only one twentieth of the size of the skin pores, making it impossible for the skin to block and invade. This is not only difficult to remove, but also brings various chemical stimuli to keratinocytes and lipid membranes, causing inflammation, thereby inducing problems such as reduced skin immunity, inducing acne, dry skin, pigmentation, damage to the skin barrier, and skin wrinkles.

[0003] Generally, the external surface charge of materials such as yellow sand, pollution, and fine dust is negatively charged due to the influence of heavy metals such as cadmium, lead, and arsenic. Therefore, research has been conducted on materials that can use electrostatic repulsion to prevent the adhesion of fine dust or remove it more easily. However, due to screening, the materials that use simple natural plant extracts are used or developed as dosage form raw materials, so the effect is minimal.

[0004] On the other hand, the skin has anionic properties due to the phospholipid components present in the stratum corneum of the skin, but there are few studies on technologies for easily removing pollutants from the skin and treating problems caused by the skin by utilizing such anions and the electrostatic force of the anions.

[0005] Therefore, the present invention is not limited to searching for materials with negative charges from simple natural extracts, but intends to process high-quality low-molecular-weight peptides from various materials by hydrolysis and separate anionic peptides. As a result, the protein was extracted from the discarded grapefruit (Citrus junos) seeds as a by-product and then hydrolyzed into low-molecular-weight peptides, and peptides with high skin absorption rate and negative charges were separated, thereby developing a material that can prevent the adhesion of fine dust such as yellow sand and pollution, and relieve skin irritation and wrinkles. Summary of the invention

[0006] Technical issues

[0007] The purpose of the present invention is to develop and provide a cosmetic composition that can prevent the adhesion of fine dust such as yellow sand and pollution and alleviate skin damage and wrinkles caused by it by separating functional low molecular weight peptides from atomic natural materials into peptides with negative charges.

[0008] Means of solving the problem

[0009] The present invention provides a cosmetic composition comprising a low molecular weight anionic peptide obtained by a process comprising the following steps: step (a), soaking grapefruit seed powder in pure water; step (b), after the soaking in the above step (a), inducing a first enzyme reaction using subtilisin derived from Bacillus subtilis; step (c), after the first enzyme reaction in the above step (b), inducing a second enzyme reaction using pepsin; step (d), after the second enzyme reaction in the above step (c), recovering a supernatant by centrifugation; step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then obtaining a supernatant by centrifugation; and step (f), after the above step (e), separating the low molecular weight anionic peptide from the supernatant obtained in the above step (e) or a solution obtained by dissolving a powder obtained by freeze-drying the supernatant in distilled water using a cation exchange resin.

[0010] The cosmetic composition of the present invention is characterized in that the molecular weight of the grapefruit seed-derived low-molecular-weight anionic peptide may be 1000 Da or less.

[0011] The cosmetic composition of the present invention is characterized in that the first enzyme reaction of the step (b) can be carried out at pH 6-8, and the second enzyme reaction of the step (c) can be carried out at pH 2-4.

[0012] The cosmetic composition of the present invention is characterized in that the first enzyme reaction of the above step (b) can be carried out at a pressure of 20 to 100 bar while maintaining a temperature of 50 to 60° C., and the second enzyme reaction of the above step (c) can be carried out at a pressure of 20 to 100 bar while maintaining a temperature of 35 to 45° C.

[0013] The cosmetic composition of the present invention is characterized in that the cosmetic composition can be used to prevent fine dust absorption.

[0014] The cosmetic composition of the present invention is characterized in that the cosmetic composition can be used to relieve skin irritation.

[0015] The cosmetic composition of the present invention is characterized in that the cosmetic composition can be used to improve skin wrinkles.

[0016] Effects of the Invention

[0017] The present invention provides a method for preparing low molecular weight peptides from grapefruit seeds and separating them according to charge, and a cosmetic composition containing the low molecular weight anionic peptides.

[0018] Furthermore, the cosmetic composition of the present invention prevents fine dust such as yellow sand and pollution from adhering to the skin, alleviates skin irritation caused by an increase in inflammatory cytokines, and alleviates skin wrinkles. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are results of confirming the surface potential of a low-molecular-weight peptide derived from yuzu seed and a low-molecular-weight anionic peptide derived from yuzu seed.

[0020] Figure 2 These are results of confirming the molecular weights of the grapefruit seed-derived low-molecular-weight peptide and the grapefruit seed-derived low-molecular-weight anionic peptide.

[0021] Figure 3 The results are to determine the efficacy of grapefruit seed-derived low molecular weight peptides and grapefruit seed-derived low molecular weight anionic peptides in inhibiting nitric oxide caused by fine dust such as yellow sand and pollution.

[0022] Figure 4 The results are to determine the efficacy of grapefruit seed-derived low molecular weight peptides and grapefruit seed-derived low molecular weight anionic peptides in inhibiting matrix metalloproteinase-1 (MMP-1).

[0023] Figure 5 The results are as follows: the effect of a cosmetic composition containing a low molecular weight anionic peptide derived from grapefruit seeds on adsorption of fine dust such as yellow sand and pollution is measured.

[0024] Figure 6 The results are to determine the efficacy of a cosmetic composition containing a grapefruit seed-derived low-molecular-weight anionic peptide in suppressing the expression of inflammatory cytokines caused by fine dust such as yellow sand and pollution. DETAILED DESCRIPTION

[0025] Citrus junos is an evergreen broad-leaved shrub of the Rutaceae family and a species of plant of the Citrus subfamily. It is mainly distributed in East Asia and was introduced to Korea during the Silla era and cultivated in the southern region. On the other hand, the seeds of the citrus junos account for 10 to 20 weight percent of the weight of the citrus pulp, are rich in active ingredients such as limonin and bioflavonoids, and contain a large amount of antioxidant substances. Therefore, they are developed into various functional materials. However, since the various active substances of the citrus seeds are mixed with a large amount of lipids, they are limited to use in oils and fats. Moreover, most of them are discarded as by-products after preparation due to their bitter taste. In addition, the citrus seeds contain not only about 20 to 30 weight percent of oil components, but also about 10 to 20 weight percent of crude protein. Despite this, there are very few efforts to utilize the citrus seeds. Therefore, the inventors extracted the protein contained in a large amount in the citrus seeds that were discarded as by-products, decomposed them into peptides that are smaller in size than proteins and are excellent in absorption rate. It is judged that new functional materials with excellent physiological activity, safety and stability can be developed by this.

[0026] The present invention provides a cosmetic composition, comprising a low molecular weight anionic peptide obtained by a process comprising the following steps: step (a), soaking grapefruit seed powder in pure water; step (b), after the soaking in the above step (a), inducing a first enzyme reaction using subtilisin derived from Bacillus subtilis; step (c), after the first enzyme reaction in the above step (b), inducing a second enzyme reaction using pepsin; step (d), after the second enzyme reaction in the above step (c), recovering a supernatant by centrifugation; step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then obtaining a supernatant by centrifugation; and step (f), after the above step (e), separating the low molecular weight anionic peptide from the supernatant obtained in the above step (e) or a solution obtained by dissolving a powder obtained by freeze-drying the supernatant in distilled water using a cation exchange resin.

[0027] Hereinafter, the preparation process of the cosmetic composition comprising the grapefruit seed-derived low-molecular-weight anionic peptide of the present invention is subdivided into each step for detailed description.

[0028] Step (a): Soak grapefruit seed powder

[0029] This step is the process of soaking the grapefruit seed powder in pure water.

[0030] After the grapefruit seeds are crushed by a grinder and a crusher to obtain grapefruit seed powder, the obtained grapefruit seed powder is soaked in pure water.

[0031] Step (b): First enzyme reaction

[0032] This step is a process of inducing the first enzyme reaction using subtilisin derived from Bacillus subtilis after the soaking in the above step (a).

[0033] In this step, the grapefruit seed powder soaked in pure water in the above step (a) is subjected to a first enzyme reaction. In this case, preferably, the first enzyme reaction is carried out under the condition of pH 6 to 8, and the pH of the first enzyme reaction solution is adjusted to 6 to 8 to best induce the enzyme reaction.

[0034] The first enzyme reaction of this step uses an enzyme derived from Bacillus subtilis, preferably, subtilisin is used as the enzyme derived from Bacillus subtilis. Specifically, the first enzyme reaction of this step can be carried out using an ultra-high pressure enzyme reactor equipped with a polymer coated with subtilisin as a protease isolated from Bacillus subtilis, preferably, at a pressure of 20 to 100 bar and a temperature of 50 to 60° C. In this case, preferably, the reaction time is 12 to 36 hours.

[0035] Step (c): Second enzyme reaction

[0036] This step is a process of inducing a second enzyme reaction using pepsin after the first enzyme reaction in the above step (b).

[0037] In this step, after the first enzyme reaction, the solution is recovered from the reactor and pepsin is post-treated to induce a second enzyme reaction. In this case, preferably, the second enzyme reaction is carried out under the condition of pH 2 to 4. The pH of the second enzyme reaction solution is adjusted to 2 to 4 to best induce the enzyme reaction.

[0038] Preferably, the second enzyme reaction of this step can be carried out in an ultra-high pressure enzyme reactor containing a polymer coated with pepsin, preferably at a pressure of 20 to 100 bar and a temperature of 35 to 45° C. In this case, preferably, the reaction time is 3 to 9 hours.

[0039] After the second enzyme reaction in this step is completed, preferably, the remaining enzyme is inactivated by boiling the solution after the secondary reaction.

[0040] Step (d): Recovering the supernatant

[0041] This step is a process of recovering the supernatant by centrifugation after the second enzyme reaction in step (c). That is, the solution after the second enzyme reaction in step (c) is centrifuged to recover the supernatant.

[0042] Step (e): Obtain supernatant after ethanol precipitation

[0043] This step is a process of adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then obtaining a supernatant by centrifugation.

[0044] In this step, ethanol precipitation is induced by adding ethanol to the supernatant recovered in the above step (d). In this case, ethanol can be added at a weight ratio of 1:1 relative to the supernatant. When ethanol is added, the precipitate is precipitated at the bottom due to the induced precipitation. Preferably, the ethanol precipitation is carried out at a refrigerated temperature.

[0045] After inducing ethanol precipitation in the above manner, the supernatant is obtained by centrifugation. In this case, it is preferred to filter the supernatant obtained by centrifugation for use, preferably, using a 0.1-0.5 μm precision filter membrane to obtain the filtrate, and after filtration, the filtrate can also be freeze-dried as needed.

[0046] Step (f): Obtaining low molecular weight anionic peptides

[0047] This step is a process of separating low molecular weight anionic peptides from the supernatant obtained in the above step (e) or the powder obtained by freeze-drying the supernatant and dissolving it in distilled water using a cation exchange resin after the above step (e).

[0048] In more detail, the low molecular weight anionic peptides of step (f) of the present invention can be obtained by separation in the following manner.

[0049] The cation exchange resin is swelled in 5 to 10 times of 1N hydrochloric acid (HCl) aqueous solution and then loaded into an open column tube. Then, preferably, after 5 to 10 times of 1M hydrochloric acid aqueous solution is flowed in for activation, 5 to 10 times of distilled water is flowed in for washing. Then, the supernatant obtained in the above step (e) or "a solution obtained by dissolving the freeze-dried powder of the supernatant in distilled water" is loaded into an open column (maximum 20 g / L resin volume), and 5 to 10 times of distilled water is continuously flowed in at an appropriate speed for elution. The eluate eluted in this way can be freeze-dried to prepare it in powder form.

[0050] After this step (f), a low molecular weight anionic peptide having a molecular weight of 1000 Da or less (a low molecular weight anionic peptide having a negative surface potential value) can be obtained. Preferably, a low molecular weight anionic peptide having a molecular weight of 500 Da or less can be obtained by additionally using size exclusion chromatography or the like.

[0051] On the other hand, preferably, the cation exchange resin used in the above step (f) can be prepared according to the following steps (a) to (e).

[0052] Step (A), agarose bead expansion

[0053] This step is a process of swelling agarose beads in a sodium chloride (NaCl) aqueous solution and then filtering.

[0054] Preferably, agarose beads as the biopolymer are swelled in 1N sodium chloride aqueous solution 5 to 10 times and then filtered.

[0055] Step (B), activation of agarose beads

[0056] This step is a process of activating the agarose filtered in the above step (A) by stirring in a sodium hydroxide (NaOH) aqueous solution. In this case, preferably, the filtered agarose is activated by stirring in a 5-10 times sodium hydroxide aqueous solution for about 30 minutes.

[0057] Step (c), preparing cation exchange resin

[0058] This step is a process of preparing a cation exchange resin by adding glycidyltrimethylammonium chloride to the agarose activated in the above step (B) and reacting the mixture. In this case, the cation exchange resin is preferably prepared by stirring and reacting the mixture at a temperature of 50 to 60° C. for 2 to 4 hours.

[0059] Step (D), washing the cation exchange resin

[0060] This step is a process of washing the cation exchange resin by adding distilled water after the above step (c). Preferably, the process of adding 5 to 10 times of distilled water and removing the supernatant after standing is performed 3 to 5 times for washing.

[0061] Step (e), pH correction

[0062] This step is a process of correcting the pH to 6.5 to 7.5 after the above step (D). On the other hand, preferably, the washing step can be added 1 to 3 times as needed, and then filtered.

[0063] In this way, by carrying out the above steps (a) to (e), the cation exchange resin for separating low molecular weight anionic peptides of the present invention is finally prepared.

[0064] In another aspect, the present invention provides a cosmetic composition comprising a low molecular weight anionic peptide obtained by a process comprising steps (a) to (f) above.

[0065] The cosmetic composition of the present invention is characterized in that the cosmetic composition can be used to prevent fine dust absorption.

[0066] The cosmetic composition of the present invention is characterized in that the cosmetic composition can be used to relieve skin irritation.

[0067] The cosmetic composition of the present invention is characterized in that the cosmetic composition can be used to improve skin wrinkles.

[0068] On the other hand, in the cosmetic composition of the present invention, the above-mentioned dust absorption prevention, skin irritation relief, or skin wrinkle improvement effect can be exerted by inhibiting the generation of nitric oxide or the expression of inflammatory cytokines caused by fine dust such as yellow sand and pollution.

[0069] In the cosmetic composition of the present invention, preferably, the low molecular weight anionic peptide of the present invention is included in an amount of 0.01 to 30.0 weight percent relative to the total weight of the cosmetic composition.

[0070] In the cosmetic composition of the present invention, the above-mentioned cosmetic composition can be any dosage form selected from the group consisting of lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) cosmetics, water-in-oil (W / O) cosmetics, ointment, foundation cream, blemish correcting powder, lipstick and scalp cosmetics.

[0071] The cosmetic composition of the present invention can be prepared into any dosage form commonly prepared in the technical field to which the present invention belongs, for example, it can be formulated into skin care water, skin care lotion, cream, essence, toner, eye wash, shampoo, conditioner, curling cream, hair spray, etc., but is not limited thereto.

[0072] On the other hand, unless otherwise defined, all technical terms used in the present invention have the following definitions, which are consistent with the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention.

[0073] In this specification, if the context does not require it, the expression "comprising" or "including" means including the proposed steps or structural elements, or a group of multiple steps or multiple structural elements, but it should be understood that it does not include the meaning of excluding other steps or structural elements, or a group of multiple steps or structural elements.

[0074] The present invention is described in more detail below through examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but also includes all modifications of the equivalent technical ideas.

[0075] Example 1: Preparation of low molecular weight peptides from grapefruit seeds

[0076] The grapefruit seeds were powdered by a pulverizer to obtain grapefruit seed powder. The grapefruit seed powder obtained above was soaked in pure water and adjusted to pH 7, and then placed in an ultra-high pressure enzyme reactor containing a polymer coated with subtilisin derived from Bacillus subtilis and maintained at 55°C under a pressure of 100 bar for 24 hours of enzymatic decomposition reaction. Then, after recovering the solution from the reactor, the pH of the solution was adjusted to 3, and after being placed in an ultra-high pressure enzyme reactor containing a polymer coated with pepsin and maintained at 40°C under a pressure of 100 bar for 6 hours of enzymatic decomposition reaction, the reaction was terminated by heating for 30 minutes to inactivate the remaining enzyme. Then, after recovering the solution from the reactor, the supernatant was obtained by centrifugation, ethanol was added at a weight ratio of 1:1 relative to the weight of the supernatant obtained above, and the supernatant after the precipitate was removed by centrifugation. Then, the filtrate was obtained by passing through a 0.2 μm precision filter membrane, and the obtained filtrate was freeze-dried to prepare grapefruit seed low molecular weight peptide powder.

[0077] Example 2: Preparation of low molecular weight anionic peptides from grapefruit seeds

[0078] (1) Preparation of cation exchange resin (affinity resin) for anionic peptide separation

[0079] In order to prepare grapefruit seed low molecular weight anionic peptides, affinity resin for separation of anionic peptides was first prepared.

[0080] Agarose beads, which are biopolymers, were swelled in a 10-fold 1N sodium chloride aqueous solution and filtered, and the filtered agarose was stirred in a 10-fold 5N sodium hydroxide aqueous solution for 30 minutes to activate. Glycidyl trimethylammonium chloride was added to the activated agarose and stirred at 55°C for 3 hours to react. Then, a washing step of adding 10-fold distilled water, leaving it to stand, and then removing the supernatant was performed 4 times, and the pH was adjusted to 7, and the above washing step was performed twice more and filtered.

[0081] (2) Preparation of grapefruit seed low molecular weight anionic peptides

[0082] The anionic peptide separation cation exchange resin prepared in Example 2-(1) was expanded in 10 times the volume of 1N HCl aqueous solution and then loaded into an open column tube. Then, 10 times the volume of 1M hydrochloric acid aqueous solution was passed through the resin, and then 10 times the volume of distilled water was passed through the resin for washing.

[0083] On the other hand, the dried grapefruit seed low molecular weight peptide powder prepared in Example 1 was dissolved in distilled water and a prescribed amount was loaded onto an open column (maximum 20 g / L resin volume), and distilled water 10 times the resin capacity was continuously flowed at an appropriate rate for elution. The eluted eluate was freeze-dried to prepare a grapefruit seed low molecular weight anionic peptide powder.

[0084] Comparative Example 1: Preparation of grapefruit seed low molecular weight anionic peptides using strong acid cation exchange resin

[0085] In Comparative Example 1, TRILITE MC-08 resin, which is a commercially available strong acidic cation exchange resin, was used to prepare grapefruit seed low molecular weight anionic peptide.

[0086] TRILITE MC-08 resin was swollen in 5 times 1M hydrochloric acid solution and then loaded into an open column tube. Then, 10 times the volume of 1M hydrochloric acid solution was flowed through the resin for activation, and then 10 times the volume of distilled water was flowed through the resin for washing.

[0087] Then, similar to Example 2, the dried grapefruit seed low molecular weight peptide powder prepared in Example 1 was dissolved in distilled water and loaded onto a specified amount of an open column (maximum 20 g / L resin volume), and then the eluent was lyophilized by continuously flowing distilled water 10 times the resin capacity at an appropriate rate to prepare the grapefruit seed low molecular weight anionic peptide powder of Comparative Example 1.

[0088] Comparative Example 2: Preparation of grapefruit seed low molecular weight anionic peptide using weakly acidic cation exchange resin

[0089] In Comparative Example 2, DIAON WK60L resin, which is a commercially available weakly acidic cation exchange resin, was used to prepare grapefruit seed low molecular weight anionic peptide.

[0090] DIAON WK60L resin was swollen in 5 times 1M hydrochloric acid aqueous solution and loaded into an open column tube. Then, 10 times the volume of 1M hydrochloric acid aqueous solution was flowed through the resin for activation, and then 10 times the volume of distilled water was flowed through the resin for washing.

[0091] Then, similar to Example 2, the dried grapefruit seed low molecular weight peptide powder prepared in Example 1 was dissolved in distilled water and loaded onto a specified amount of an open column (maximum 20 g / L resin volume), and then the eluate was lyophilized by continuously flowing distilled water 10 times the resin capacity at an appropriate rate to prepare the grapefruit seed low molecular weight anionic peptide powder of Comparative Example 2.

[0092] Experimental Example 1: Measuring surface potential

[0093] In this experimental example, an experiment was conducted to measure the surface potential of the above-mentioned Examples 1 and 2, Comparative Examples 1 and 2. In order to analyze the surface charge, the surface charge of each peptide was confirmed using a zeta potential analyzer, which is a measurement device for dynamic light scattering and electrophoretic light scattering, and the measured values ​​were measured three times to obtain an average value.

[0094] Figure 1 The results of confirming the surface potential of the grapefruit seed low molecular weight peptide (Example 1) and the grapefruit seed low molecular weight anionic peptide (Example 2) are shown. Figure 1 As shown, the zeta potential of Example 1 was measured to be -0.84 mV, and the zeta potential of Example 2 was measured to be -8.72 mV. It can be seen that the low molecular weight anionic peptides were well separated in Example 2 of the present invention using a cation exchange resin (affinity resin).

[0095] In addition, the ZETA potential of Comparative Example 1 was measured to be -3.25 mV, and the ZETA potential of Comparative Example 2 was measured to be -4.82 mV, confirming that the cation exchange resin (affinity resin) for anionic peptide separation prepared in Example 2 has a higher separation ability for low molecular weight anionic peptides than the commercially available strong acid and weak acid cation exchange resins of Comparative Examples 1 and 2.

[0096] Experimental Example 2: Determination of Molecular Weight

[0097] In this experimental example, the molecular weight measurement experiment of the above-mentioned Example 1 and Example 2 was carried out. In order to measure the molecular weight, a MALDI-TOF mass spectrometer (MALDI-TOF MS, Matrix-assisted laser desorption-ionization time-of-flight mass spectrometer) was used.

[0098] After Example 1 and Example 2 were dissolved in a 50% (v / v) methanol / water mixture, 1 μl was taken and mixed with 1 μl of sinapinic acid (SA) substrate (matrix) (50% (v / v) 0.1% trifluoroacetic acid (TFA) acetonitrile / 50% (v / v) water). Then, the above mixture was spotted onto a stainless steel MALDI plate and dried at room temperature. The analysis was performed using a Microflex LRF MALDI-TOF mass spectrometer and a Bruker UltrafleXtreme (Bruker UltrafleXtreme, Bruker Daltonics, Bremen, Germany). The intensity of each ion was calculated by diffusing the first isotope peak area to the third isotope peak area. The spectrum was acquired and processed using FlexAnalysis software (ver.3.3, Bruker Daltonics, Bremen, Germany), and the results are shown as follows. Figure 2 shown.

[0099] Figure 2The results of confirming the molecular weights of grapefruit seed low molecular weight peptides and low molecular weight anionic peptides are shown. The molecular weight distribution of Examples 1 and 2 is below 1000 Da, and more specifically, most of the molecular weight distribution is below 500 Da. This indicates that Examples 1 and 2 are well hydrolyzed into low molecular weights.

[0100] Experimental Example 3: Confirmation of the inhibition rate of nitric oxide caused by fine dust

[0101] In this experimental example, an experiment was conducted to confirm the inhibitory effect of the above-mentioned Examples 1 and 2 on nitric oxide generated by the stimulation of fine dust.

[0102] RAW 264.7 cells, which are mouse-derived macrophages, were cultured at 1×10 5 After cells / well were dispensed into 96-well plates, they were stabilized in a cell culture incubator at 37°C and 5% CO2 for one day, and then treated with 200μg / ml dust. After each sample was diluted to a concentration of 200μg / ml, 5μM of nordihydroguaiaretic acid (NDGA) was treated as a positive control group and reacted for 24 hours. An equal amount of 100μl of Griess reagent was added to 100μl of the separated culture supernatant and reacted at room temperature, and then the absorbance was measured at a wavelength of 540nm using a microplate reader. The concentration of nitric oxide was calculated by comparing it with a standard straight line obtained using sodium nitrite (NaNO2).

[0103] Figure 3 The results of measuring the efficacy of low molecular weight peptides and low molecular weight anionic peptides derived from grapefruit seeds in inhibiting nitric oxide caused by fine dust such as yellow sand and pollution are shown. Figure 3 As shown, it was confirmed that the activity of suppressing nitric oxide was higher when Example 2 was treated than when NDGA or Example 1 was treated. That is, although Example 1 (low-molecular-weight peptide from grapefruit seed) also showed the effect of suppressing nitric oxide, the activity of suppressing nitric oxide production was more excellent in Example 2 (low-molecular-weight anionic peptide from grapefruit seed) based on charge separation.

[0104] Experimental Example 4: Determination of the inhibitory effect of MMP-1 production

[0105] In this experimental example, an experiment was conducted to confirm the effect of the above-mentioned Example 1 and Example 2 on suppressing the production of MMP-1 (matrix metalloproteinase-1).

[0106] Human dermal fibroblasts (HDFn) were cultured at 5×104 After cells / well were dispensed into 24-well culture plates, the cells were stabilized in a cell culture incubator at 37°C and 5% CO2 for one day and then injected at 1 J / cm 2 Treated with long-wave ultraviolet light (UVA). Each sample was treated with a medium supplemented with 2% fetal bovine serum (FBS) and diluted to different concentrations. Adenosine (a functional wrinkle notification material) was treated at a concentration of 400 μM as a positive control group and reacted for 48 hours. The supernatant was used to confirm the expression of MMP-1 using the R&D systems kit (DY901), and the particles were treated with MTT solution (MTSolution) to confirm cytotoxicity.

[0107] After the MMP-1 ELISA assay, the absorbance was measured at a wavelength of 450 nm to calculate the amount of MMP-1 produced, and then the MMP-1 expression inhibition rate (%) was calculated by comparison with the control group. The calculation formula is as follows.

[0108] Mathematical formula 1

[0109]

[0110] Figure 4 The results of measuring the MMP-1 inhibitory efficacy of grapefruit seed-derived low molecular weight peptides and low molecular weight anionic peptides are shown, as Figure 4 As shown, it was confirmed that the activity of inhibiting the production of MMP-1 was superior when Example 2 was treated compared to the case of Example 1. That is, although Example 1 (low-molecular-weight peptide from citron seed) also showed the activity of inhibiting the production of MMP-1, the activity of inhibiting the production of MMP-1 was superior in Example 2 (low-molecular-weight anionic peptide from citron seed) by charge separation.

[0111] Preparation Example 1: Preparation of dosage form using Example 1 and Example 2

[0112] In this preparation example, the cosmetic compositions of Preparation Example 1 and Preparation Example 2 using the peptide of Example 1 (grapefruit seed low molecular weight peptide) or Example 2 (grapefruit seed low molecular weight anionic peptide) and Comparative Example 3 not using both Example 1 and Example 2 were prepared with the compositions shown in Table 1 below.

[0113] Table 1

[0114]

[0115]

[0116] Experimental Example 5: Determination of anti-dust adsorption effect

[0117] In this experimental example, the anti-dust adsorption effect of the cosmetic compositions of Preparation Example 1 (using Example 1), Preparation Example 2 (using Example 2), and Comparative Example 3 (not using both Example 1 and Example 2) was confirmed. For this purpose, carbon black having a size and surface potential similar to those of dust particles was used.

[0118] In order to achieve the state of fine dust floating in the atmosphere, a fine dust floating chamber equipped with paddles was used to evenly disperse an appropriate amount of carbon black. The adsorption effect test method was to apply Comparative Example 3, Preparation Example 1 and Preparation Example 2 to the divided areas on the forearm, and after natural drying, use a high-resolution digital camera and Folliscope (magnified photos of the skin surface) to take pictures and use an image analysis program to analyze the brightness value of the test area to obtain the change before and after the fine dust adsorption ( Figure 5 On the other hand, the fine dust adsorption amount indicates that the fine dust adsorption decreases with the increase of the lightness value. When the adsorption amount (Δ) of the test product treatment group is statistically significantly (p<0.05) reduced relative to the adsorption amount (Δ) of the non-treatment group, it is judged to have an anti-fine dust adsorption effect.

[0119] The improvement rate (%) of Comparative Example 3, Preparation Example 1 and Preparation Example 2 relative to the untreated group was evaluated by analyzing the dust adsorption amount. The results are shown in Table 2 and Figure 5 shown.

[0120] Mathematical formula 2

[0121]

[0122] Table 2

[0123]

[0124] As shown in Table 2 and Figure 5 As shown, the lightness value of Preparation Example 2 using grapefruit seed low molecular weight anionic peptide (Example 2) was 68.1%, and the improvement rate was 8.3%. It was confirmed that the dust adsorption amount was significantly improved compared with the cosmetic compositions of Preparation Example 1 (using Example 1) and Comparative Example 3 (control group).

[0125] Experimental Example 6: Confirmation of the expression of inflammatory cytokines caused by fine dust

[0126] In the experimental examples, the cosmetic compositions of Preparation Example 1 (using Example 1), Preparation Example 2 (using Example 2) and Comparative Example 3 (not using both Example 1 and Example 2) were confirmed to have an effect of inhibiting the expression of inflammatory cytokines generated by fine dust stimulation.

[0127] For the experiment, 30 subjects (20-35 years old women) were used as the subjects, and the skin surface cytokines were obtained after applying sebum tape (sebutape) on the forearm before applying the dust. This process was carried out 5 times in total and each was placed in a glass vial containing 1ml of phosphate buffer solution (PBS buffer), and the cytokine content was measured after an ultrasonic (sonicator) for 1 hour.

[0128] Then, the fine dust and mineral oil were mixed in a ratio of 1:1 and then treated on the forearm, and the sebutape patch was applied as in Comparative Example 3, Preparation Example 1, and Preparation Example 2. After 24 hours, the patch was removed, and cytokines were obtained in the same manner as before the above-mentioned application. Cytokines were quantified using an enzyme-linked immunosorbent assay kit (ELISA kit), and total protein was quantified using a protein quantification kit (kit). The cytokine content was corrected using the total protein content to analyze the results ( Figure 6 ).

[0129] like Figure 6 As shown, it was confirmed that the cosmetic compositions of Preparation Example 1 using the low molecular weight peptide derived from citrus seeds (Example 1) and Preparation Example 2 using the low molecular weight anionic peptide derived from citrus seeds (Example 2) significantly reduced the expression levels of inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β)) caused by fine dust compared to Comparative Example 3, and in particular, Preparation Example 2 using the low molecular weight anionic peptide derived from citrus seeds (Example 2) was confirmed to exhibit a more excellent effect. That is, it was found that when the low molecular weight anionic peptide derived from citrus seeds of the present invention is used, a cosmetic composition having an excellent skin irritation alleviating effect can be prepared.

[0130] Experimental Example 7: Measurement of wrinkle improvement effect

[0131] In this experimental example, the skin wrinkle improving effect of the cosmetic compositions of Preparation Example 1 (using Example 1), Preparation Example 2 (using Example 2), and Comparative Example 3 (not using both Example 1 and Example 2) was confirmed.

[0132] Therefore, 30 subjects (women aged 20-35 years old) were used as the subjects, and the wrinkle improvement effects before and after 10 weeks of use of the cosmetic compositions of Preparation Example 1 (using Example 1), Preparation Example 2 (using Example 2) and Comparative Example 3 (not using both Example 1 and Example 2) were evaluated by instrumental measurement (cutometer SEM575, C+K Electronic, Germany).

[0133] Table 3 below shows the results of the measurement of the skin wrinkle improvement effect. The experiment was conducted by applying Preparation Example 1 to the face of Group A, Preparation Example 2 to the face of Group B, and Comparative Example 3 to the face of Group C.

[0134] Table 3

[0135]

[0136] As shown in Table 3, it was confirmed that the cosmetic compositions of Preparation Example 1 using the grapefruit seed low molecular weight peptide (Example 1) and Preparation Example 2 using the grapefruit seed low molecular weight anionic peptide (Example 2) showed excellent wrinkle improvement effects compared to Comparative Example 3, and in particular, it was confirmed that a more excellent effect was exhibited in Preparation Example 2 using the grapefruit seed low molecular weight anionic peptide (Example 2). That is, it was found that in the case of the grapefruit seed-derived low molecular weight anionic peptide of the present invention, a cosmetic composition having an excellent skin wrinkle improvement effect can be prepared.

[0137] As described above, the present invention has confirmed that the low-molecular-weight anionic peptide derived from grapefruit seeds and the cosmetic composition containing the same are effective in fine dust adsorption, skin irritation relief, and skin wrinkle improvement. That is, the surface potential of the low-molecular-weight anionic peptide derived from grapefruit seeds of the present invention shows a negative charge, and can effectively reduce fine dust adsorption by electrostatic repulsion with fine dust. The present invention contains the low-molecular-weight anionic peptide derived from grapefruit seeds, and therefore, can prepare a cosmetic composition with excellent effects of alleviating skin irritation caused by an increase in inflammatory cytokines caused by fine dust and improving skin wrinkles.

Claims

1. A cosmetic composition, characterized in that The invention comprises a low molecular weight anionic peptide obtained by a process comprising the steps of: Step (a), soaking grapefruit seed powder in pure water; Step (b), after the soaking in the above step (a), using subtilisin derived from Bacillus subtilis to induce a first enzyme reaction; Step (c), after the first enzyme reaction in the above step (b), using pepsin to induce a second enzyme reaction; Step (d), after the second enzyme reaction in step (c), recovering the supernatant by centrifugation; Step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then obtaining a supernatant by centrifugation; as well as Step (f), after the above step (e), using a cation exchange resin to separate the low molecular weight anionic peptides from the supernatant obtained in the above step (e) or the powder obtained by freeze-drying the supernatant and dissolving it in distilled water.

2. The cosmetic composition according to claim 1, characterized in that The molecular weight of the grapefruit seed-derived low-molecular-weight anionic peptide is less than 1000 Da.

3. The cosmetic composition according to claim 1, characterized in that The first enzyme reaction of step (b) is carried out at pH 6 to 8. The second enzyme reaction in the above step (c) is carried out at pH 2-4.

4. The cosmetic composition according to claim 1, characterized in that The first enzyme reaction of step (b) is carried out at a pressure of 20 to 100 bar and a temperature of 50 to 60° C. The second enzyme reaction in step (c) is carried out at a pressure of 20 to 100 bar and a temperature of 35 to 45°C.

5. The cosmetic composition according to claim 1, characterized in that The above-mentioned cosmetic composition is used for preventing fine dust absorption.

6. The cosmetic composition according to claim 1, characterized in that The above-mentioned cosmetic composition is used to relieve skin irritation.

7. The cosmetic composition according to claim 1, characterized in that The above cosmetic composition is used for improving skin wrinkles.

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