Soy oligopeptide for cosmetics, and preparation method and application thereof

Abstract

The present application relates to a kind of soybean oligopeptide for cosmetics and its preparation method and application.The soybean oligopeptide is extracted from soybean, and the soybean oligopeptide is prepared into soybean oligopeptide composition in specific proportion, has significant synergistic antioxidant, anti-inflammatory, repair and whitening effect, and is used to prepare into cosmetics;The prepared cosmetic is safe to human skin, has good anti-inflammatory, repair, whitening and antioxidant effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of daily cosmetics, and particularly relates to a soybean oligopeptide for cosmetics, a preparation method and application thereof. BACKGROUND

[0002] In recent years, natural cosmetics have gradually become one of the development directions of cosmetics, and the selection of natural raw materials has become a hot spot in the cosmetics industry. Soybean is the most widely known plant protein source, and protein accounts for 35-40% of the dry weight of soybean. In addition to protein, soybean also contains fat, vitamins, minerals, carbohydrates, isoflavones, saponins and other substances with health functions.

[0003] Soybean is widely used in the preparation of cosmetics, mainly using the rich active ingredients in soybean, such as soybean isoflavones, soybean peptides, soybean saponins and soybean proteins. These components have various effects on skin care.

[0004] Soybean peptides are small molecular peptides produced by hydrolysis of soybean protein, which have good moisturizing and repairing effects. It can promote the synthesis of collagen, increase the firmness of the skin, and also improve the barrier function of the skin and enhance the resistance of the skin. Soybean protein has good emulsifying and film-forming properties. It can help improve the texture of cosmetics, increase the stability of products, and also provide certain moisturizing effect for the skin.

[0005] Oligopeptide is a short-chain protein molecule composed of 2 to 20 amino acids connected by peptide bonds. It is a short-chain peptide produced during protein hydrolysis or synthesis, which is usually more easily absorbed and utilized by the skin than long-chain proteins such as collagen and keratin.

[0006] Oligopeptide has a molecular weight much smaller than protein, making it easier to penetrate the stratum corneum and be absorbed and utilized by the skin. Oligopeptide retains the activity of amino acids and can better participate in various physiological activities of cells. Different sequences of oligopeptides have different biological activities, such as anti-aging, repair, and moisturizing.

[0007] Oligopeptide can promote the production of collagen and elastin, increase the elasticity and firmness of the skin, and reduce the formation of wrinkles and fine lines. Oligopeptide has good moisturizing properties and can help the skin lock in moisture, improving dry and rough skin. Oligopeptide can promote cell repair and regeneration, accelerate wound healing, and improve damaged skin. Oligopeptide can help the skin better absorb other skincare ingredients, improving the overall effect of skincare products.

[0008] Oligopeptides are increasingly used in cosmetics due to their small molecular weight and ability to penetrate the skin. They can promote skin repair, anti-aging, improve skin color, and moisturize, and are particularly suitable for anti-aging, repair, and sensitive skin care. They are one of the ingredients that cannot be ignored in modern skin care products. SUMMARY

[0009] To solve the problems in the prior art, the present application provides a salt mud cleaning and moisturizing composition, a preparation method and application thereof.

[0010] To this end, the present application adopts the following technical solutions:

[0011] In one aspect, the present application relates to a soybean oligopeptide composition, characterized in that it comprises soybean oligopeptide 1 and soybean oligopeptide 2.

[0012] The number of amino acids in the amino acid sequence of the soybean oligopeptide 1 and the soybean oligopeptide 2 is 3 to 7;

[0013] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 1 is 4 to 7;

[0014] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 1 is 5, 6, or 7;

[0015] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 1 is 6 or 7;

[0016] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 1 is 7;

[0017] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 2 is 3 to 6;

[0018] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 2 is 4, 5, or 6;

[0019] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 2 is 5 or 6;

[0020] Preferably, the number of amino acids in the amino acid sequence of the soybean oligopeptide 2 is 6.

[0021] In another aspect, the present application relates to a soybean oligopeptide composition, characterized in that the mass ratio of the soybean oligopeptide 1 to the soybean oligopeptide 2 is (1-3):(3-1);

[0022] Preferably, the mass ratio of the soybean oligopeptide 1 to the soybean oligopeptide 2 is 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, or 3:2;

[0023] Preferably, the mass ratio of the soybean oligopeptide 1 to the soybean oligopeptide 2 is 2:3.

[0024] In another aspect, the present application relates to a soybean oligopeptide composition, wherein the soybean oligopeptide 1 is:

[0025] (1) an oligopeptide having an amino acid sequence of LQVYKFT (SEQ ID No: 1); and / or

[0026] (2) an oligopeptide having an amino acid sequence with at least 57.14 % homology to the amino acid sequence of LQVYKFT (SEQ ID No: 1) and having the same or similar function.

[0027] Preferably, the amino acid sequence of the soybean oligopeptide 1 is LQVYKFT (SEQ ID No: 1).

[0028] Preferably, the amino acid sequence of the soybean oligopeptide 1 is an oligopeptide having at least 4 amino acids identical to the amino acid sequence of LQVYKFT (SEQ ID No: 1) and having the same or similar function.

[0029] Preferably, the amino acid sequence of the soybean oligopeptide 1 is an oligopeptide having at least 5 amino acids identical to the amino acid sequence of LQVYKFT (SEQ ID No: 1) and having the same or similar function.

[0030] Preferably, the amino acid sequence of the soybean oligopeptide 1 is an oligopeptide having at least 6 amino acids identical to the amino acid sequence of LQVYKFT (SEQ ID No: 1) and having the same or similar function.

[0031] In another aspect, the present application relates to a soybean oligopeptide composition, wherein the soybean oligopeptide 2 is:

[0032] (1) an oligopeptide having an amino acid sequence of QLSRMG (SEQ ID No: 2); and / or

[0033] (2) an oligopeptide having an amino acid sequence with at least 66.67 % homology to the amino acid sequence of QLSRMG (SEQ ID No: 2) and having the same or similar function.

[0034] Preferably, the amino acid sequence of the soybean oligopeptide 2 is QLSRMG (SEQ ID No: 2).

[0035] Preferably, the amino acid sequence of the soybean oligopeptide 2 is an oligopeptide having at least 3 amino acids identical to the amino acid sequence of QLSRMG (SEQ ID No: 2) and having the same or similar function.

[0036] Preferably, the amino acid sequence of the soybean oligopeptide 2 is an oligopeptide having at least 4 amino acids identical to the amino acid sequence of QLSRMG (SEQ ID No: 2) and having the same or similar function.

[0037] Preferably, the amino acid sequence of the soybean oligopeptide 2 is an oligopeptide having at least 5 amino acids identical to the amino acid sequence of QLSRMG (SEQ ID No: 2) and having the same or similar function.

[0038] The soybean oligopeptides shown in SEQ ID No: 1 and SEQ ID No: 2 were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. and stored for future use.

[0039] In another aspect, the present application relates to a soybean oligopeptide preparation method comprising the following steps:

[0040] (1) Extraction of soybean protein: 20 g of soybean was weighed, crushed through a 60-mesh sieve to obtain soybean powder, 40 mL of plant protein extraction solution was added, and homogenate was prepared by grinding;

[0041] (2) Tris balanced phenol saturated solution layering: supplement the protein extraction solution to 100 mL, mix well, add 100 mL of Tris balanced phenol saturated solution with pH of 8.0, mix at 4 ℃ for 30 min, and mix multiple times; centrifuge at 10000 g for 15 min at 4 ℃, collect the upper liquid, add 5 times the volume of pre-cooled 0.1 M ammonium acetate-methanol solution, and precipitate the protein at -20 ℃ overnight;

[0042] (3) Desalination: centrifuge at 12000 g for 10 min at 4 ℃, mix well after adding water to the precipitate, inject into a dialysis bag, and dialyze in pure water for 24 to 48 h, replace the pure water 4 to 6 times during the period, until the water outside the dialysis bag is colorless and the conductivity is close to that of pure water, then centrifuge at 12000 g for 10 min to obtain the desalted protein precipitate;

[0043] (4) Methanol-acetone washing of protein precipitate: add 5 times the volume of pre-cooled methanol to wash the above protein precipitate, and mix slightly; replace methanol with acetone and repeat the above steps twice to completely remove methanol; centrifuge at 12000 g for 10 min at 4 ℃, collect the precipitate; freeze-dry the precipitate, dissolve the dried powder in 100 mL of sterile ultrapure water, vortex wash, then centrifuge at 12000 g for 10 min at room temperature, and collect the supernatant;

[0044] (5) Ultrafiltration and freeze-drying: the supernatant was transferred to a 10 KD ultrafiltration centrifuge tube, centrifuged at 12000 g for 15 min at 4 DEG C, and the filtrate and the filtrate were collected respectively. The filtrate was transferred to a 1 KD ultrafiltration centrifuge tube, centrifuged at 12000 g for 15 min at 4 DEG C, and the filtrate and the filtrate were collected respectively. The filtrate was the crude soybean oligopeptide extract, which was freeze-dried to obtain the finished soybean oligopeptide, and stored at -20 DEG C for standby;

[0045] (6) Soybean oligopeptide sequencing: for the high-throughput sequencing of the prepared soybean oligopeptide, the peptide sequences with high sequencing result score, high reliability and high frequency were screened, and the soybean oligopeptide 1 with the amino acid sequence LQVYKFT (SEQ ID No: 1) and the soybean oligopeptide 2 with the amino acid sequence QLSRMG (SEQ ID No: 2) were obtained.

[0046] On the other hand, the present application relates to a composition with anti-inflammatory, repair, whitening and antioxidant effects, characterized by comprising the following components in mass percentage:

[0047] The soybean oligopeptide composition according to any of the present application 10.00 %, 1,3-propanediol 30.00 %, glycerol 45.00 %, preservative 0.05 % and water balance;

[0048] The preservative is selected from one or more of hydroxybenzoate, hydroxybenzylate, phenoxyethanol, chlorphenesin, bis (hydroxymethyl) imidazole alkyl urea.

[0049] Further, the composition with anti-inflammatory, repair, whitening and antioxidant effects is prepared by the following method:

[0050] ① According to the formula, soybean oligopeptide 1 and soybean oligopeptide 2 were weighed and mixed uniformly to prepare a soybean oligopeptide composition for standby;

[0051] ② According to the formula, 1,3-propanediol, glycerol, preservative and appropriate amount of water were mixed uniformly, heated to 45 DEG C under stirring condition, and completely dissolved to obtain I phase;

[0052] ③ I phase and appropriate amount of water were added to the emulsifying pot, the stirrer and high-speed homogenizer were started, and homogenized at 50 DEG C until uniform;

[0053] ④ The homogenizer was turned off and the temperature was lowered, when the emulsifying pot was cooled to room temperature, the soybean oligopeptide composition was added to the emulsifying pot, and the water was added to 100 % and stirred until uniform to obtain the finished product;

[0054] The finished product is a cosmetic water.

[0055] In another aspect, the present application relates to a composition with anti-inflammatory, repair, whitening and antioxidant efficacy, characterized in that it comprises the following components in the following weight percentage:

[0056] The soybean oligopeptide composition according to any of the present application 8.00 %, 1,3-butanediol 6.00 %, cetyl alcohol octanoate 6.00 %, 1,2-hexanediol 7.00 %, glycerol 7.00 %, dimethicone 3.50 %, polyglyceryl-3 methyl glucose distearate 1.20 %, cetyl stearyl alcohol 1.50 %, hydroxypropyl starch phosphate 2.00 %, sodium stearoyl glutamate 1.50 %, panthenol 3.00 %, hydrogenated lecithin 1.50 %, tocopheryl acetate 0.10 %, glyceryl octanoate 0.10 %, stearic acid 0.50 %, palmitic acid 0.50 %, disodium EDTA 1.50 %, ethylhexylglycerin 3.50 %, polysorbate-20 1.50 %, sorbitan isostearate 0.50 %, myristic acid 0.10 %, 7-dehydrocholesterol 0.10 %, gluconolactone 1.00 %, lauric acid 1.00 %, tocopherol 0.50 %, preservative 0.05 % and water balance;

[0057] The preservative is selected from one or more of methylparaben, propylparaben, phenoxyethanol, chlorphenesin, bis(hydroxymethyl)imidazolidinyl urea.

[0058] Further, the composition with anti-inflammatory, repair, whitening and antioxidant efficacy is prepared by the following method:

[0059] ①According to the formula, soybean oligopeptide 1 and soybean oligopeptide 2 are weighed and mixed uniformly to prepare soybean oligopeptide composition for standby;

[0060] ②According to the formula, cetyl alcohol octanoate, 1,2-hexanediol, glycerol, dimethicone, polyglyceryl-3 methyl glucose distearate, cetyl stearyl alcohol, hydroxypropyl starch phosphate, panthenol, polysorbate-20 and sorbitan isostearate are weighed and mixed uniformly, heated to 70℃ under stirring condition to completely dissolve, to obtain phase I;

[0061] ③Phase I and appropriate amount of water are added to the emulsifying pot, the stirrer and high-speed homogenizer are started, and homogenized at 65℃ until uniform;

[0062] IV. Turn off the heating and start to cool down, when the emulsifying pot is cooled to 45℃, add sodium stearoyl glutamate, hydrogenated lecithin, tocopherol acetate, glyceryl octanoate, stearic acid, palmitic acid, disodium EDTA, myristic acid, 7-dehydrocholesterol, gluconolactone, lauric acid, tocopherol, preservative and appropriate amount of water into the emulsifying pot, homogenize for 5min, when the temperature of the emulsifying pot is cooled to 35℃, add ethylhexylglycerin into the emulsifying pot, homogenize for 5min;

[0063] V. When the emulsifying pot is cooled to room temperature, add the soybean oligopeptide composition into the emulsifying pot, and add water to 100%, stir until uniform, to obtain the finished product;

[0064] The finished product is a cream.

[0065] In another aspect, the present application relates to the use of the soybean oligopeptide composition according to any one of the present application in the preparation of a cosmetic product having anti-inflammatory, repair, whitening and antioxidant effects;

[0066] Further, the cosmetic product includes but is not limited to facial cleanser, jelly, emulsion, lotion, cream, eye cream, mask, essence, aerosol or spray;

[0067] Preferably, the cosmetic product is a lotion or a cream.

[0068] The present application has the following advantages and beneficial effects over the prior art:

[0069] The soybean oligopeptide composition provided by the present application is safe and non-toxic to cells, can significantly promote the proliferation of human keratinocytes and inhibit the activity of tyrosinase, has potential repair and whitening effects, and can be applied in various fields such as cosmetic products and beauty products. At the same time, it has obvious effects in whitening and antioxidant. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 . Effect of soybean oligopeptide and its composition on LO2 cell activity.

[0071] Figure 2 . Effect of soybean oligopeptide and its composition on LPS-induced NO release.

[0072] Figure 3 . Effect of soybean oligopeptide composition on tyrosinase activity.

[0073] Figure 4 . ABTS radical scavenging capacity of soybean oligopeptide and its composition.

[0074] Figure 5 . Evaluation of the effect of soybean oligopeptide and its composition on keratinocyte proliferation.

[0075] Figure 6 . Evaluation of efficacy of different samples on skin barrier repair. DETAILED DESCRIPTION

[0076] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The raw materials, reagents or devices used in the following examples are commercially available or can be obtained by known methods unless otherwise specified. The experimental methods without specific conditions are usually carried out according to the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the parts and percentages are parts by weight and weight percentages.

[0077] Example 1 Preparation of soybean oligopeptide

[0078] The preparation of soybean oligopeptide includes the following steps:

[0079] (1) Extraction of soybean protein: soybean for cosmetics (GLYCINE MAX) was weighed, crushed to pass through a 60-mesh sieve to obtain soybean powder, and 40 mL of plant protein extraction solution (P1258, purchased from Beijing Puli Lei Gene Technology Co., Ltd.) was added for homogenization grinding;

[0080] (2) Tris balanced phenol saturated solution layering: supplement the protein extraction solution to 100 mL, mix well, add 100 mL of Tris balanced phenol saturated solution with pH 8.0, mix at 4 ℃ for 30 min, and mix several times; centrifuge at 10000 g for 15 min at 4 ℃, collect the upper liquid, add 5 times the volume of pre-cooled 0.1 M ammonium acetate-methanol solution, and precipitate the protein at -20 ℃ overnight;

[0081] (3) Desalination: centrifuge at 12000 g for 10 min at 4 ℃, mix well with water in the precipitate, inject into a dialysis bag, and dialyze in pure water for 24 to 48 h, replace the pure water 4 to 6 times during the period, until the water outside the dialysis bag is colorless and the conductivity is close to that of pure water, then centrifuge at 12000 g for 10 min to obtain the desalted protein precipitate;

[0082] (4) Methanol-acetone washing of protein precipitate: add 5 times the volume of pre-cooled methanol to wash the above protein precipitate, mix slightly; replace methanol with acetone and repeat the above steps twice to completely remove methanol; centrifuge at 12000 g for 10 min at 4 ℃, collect the precipitate; freeze-dry the precipitate, dissolve the dried powder in 100 mL of sterile ultrapure water, vortex wash, then centrifuge at 12000 g for 10 min at room temperature, and collect the supernatant;

[0083] (5) Ultrafiltration and lyophilization: Transfer the supernatant to a 10 KD ultrafiltration centrifuge tube, centrifuge at 12000 g for 15 min at 4 ℃, and collect the filter membrane protein liquid and the filtrate, respectively. Transfer the filtrate to a 1 KD ultrafiltration centrifuge tube, centrifuge at 12000 g for 15 min at 4 ℃, and collect the filter membrane protein liquid and the filtrate, respectively. The filtrate is the crude soybean oligopeptide extract, which is freeze-dried to obtain the finished soybean oligopeptide, which is stored at -20 ℃ for standby;

[0084] (6) High-throughput sequencing of the prepared soybean oligopeptide to screen the peptide sequences with high sequencing result score, high reliability and highest frequency: LQVYKFT (SEQ ID No: 1) and QLSRMG (SEQ ID No: 2), which are synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. and stored for standby.

[0085] A small amount of soybean oligopeptide 1 is prepared by solid-phase synthesis method for laboratory test, and the preparation method comprises:

[0086] (1) Resin swelling

[0087] Weigh Rink Amide AM resin (200-400 mesh), swell in DMF for 15 minutes, and wash with DMF;

[0088] (2) Deprotection

[0089] Treat the Fmoc protecting group on the resin with a piperidine / DMF (v / v 2:8) solution, treat for 2 times for 5 minutes to remove the Fmoc group, and wash the resin with DMF multiple times;

[0090] (3) Loading the first amino acid

[0091] Mix the first amino acid (Fmoc-Thr, 2 equivalents) with an activating agent (HATU, 2 equivalents) and a conjugating agent (DIPEA, 4 equivalents), add to the swollen resin, stir at room temperature for 2 hours, wash with a piperidine / DMF (v / v 2:8) solution for 20 minutes to remove the Fmoc group, and then wash the resin with DMF multiple times to remove unreacted reagents and by-products;

[0092] (4) Stepwise extension of amino acids

[0093] Mix the next amino acid (Fmoc-Phe, 2 equivalents) with an activating agent (HATU, 2 equivalents) and a conjugating agent (DIPEA, 4 equivalents), add to the resin for coupling reaction, and the reaction time is 2 hours. After coupling of each amino acid, repeat the deprotection and washing steps. Add each amino acid in SEQ ID No: 1 in turn, and repeat the above steps until all the amino acids are synthesized.

[0094] (5) Final deprotection and cleavage

[0095] After the last amino acid was synthesized, the last deprotection was performed with a piperidine / DMF (v / v 2:8) solution, the resin was washed with DMF several times, and the resin was treated with a cleavage solution (TFA:TIPS:water = 95:5:5) at room temperature for 2 hours to cleave the oligopeptide and remove the side chain protecting groups. The cleavage mixture was collected, the solvent was removed under vacuum, and the crude peptide was dissolved in 0.1% TFA in water and lyophilized;

[0096] (6) Chromatography and purification

[0097] The synthesized oligopeptide was purified using HPLC, the relevant fractions were collected, lyophilized, and analyzed by LC-MS, MALDI-TOF MS, and analytical HPLC.

[0098] A small amount of soy oligopeptide 2 was prepared for laboratory testing using solid-phase synthesis, and the preparation method included:

[0099] (1) Resin swelling

[0100] Rink Amide AM resin (200-400 mesh) was weighed and swelled in DMF for 15 minutes and washed with DMF;

[0101] (2) Deprotection

[0102] The Fmoc protecting group on the resin was treated with a piperidine / DMF (v / v 2:8) solution, and 2 5-minute treatments were performed to remove the Fmoc group. The resin was washed with DMF several times;

[0103] (3) Loading the first amino acid

[0104] The first amino acid (Fmoc-Gly, 2 equivalents) was mixed with an activating reagent (HATU, 2 equivalents) and a conjugating reagent (DIPEA, 4 equivalents) and added to the swelled resin. The reaction was stirred at room temperature for 2 hours, and a piperidine / DMF (v / v 2:8) solution was used to wash for 20 minutes to remove the Fmoc group. The resin was then washed with DMF several times to remove unreacted reagents and byproducts;

[0105] (4) Stepwise extension of amino acids

[0106] The next amino acid (Fmoc-Met, 2 equivalents) was mixed with an activating reagent (HATU, 2 equivalents) and a conjugating reagent (DIPEA, 4 equivalents) and added to the resin for coupling. After each amino acid coupling, the deprotection and washing steps were repeated. Each amino acid in SEQ ID No: 2 was added in turn, and the above steps were repeated until all the amino acids were synthesized.

[0107] (5) Final deprotection and cleavage

[0108] After the last amino acid was synthesized, the last deprotection was performed with a piperidine / DMF (v / v 2:8) solution, the resin was washed with DMF several times, and the resin was treated with a cleavage solution (TFA:TIPS:water = 95:5:5) at room temperature for 2 hours to cleave the oligopeptide and remove the side chain protecting groups. The cleavage mixture was collected, the solvent was removed under vacuum, and the crude peptide was dissolved in 0.1% TFA in water and lyophilized;

[0109] (6) Chromatography and purification

[0110] The synthesized oligopeptides were purified using HPLC, the relevant fractions were collected, lyophilized, and analyzed by LC-MS, MALDI-TOF MS, and analytical HPLC.

[0111] Example Two Evaluation of the Cytotoxicity of Soybean Oligopeptides and Compositions Thereof

[0112] 1. Experimental samples

[0113] Soybean oligopeptide 1 (BO1), soybean oligopeptide 2 (BO2), and soybean oligopeptide compositions were dissolved in RPMI-1640 medium (containing 10% FBS, 1% PS) to prepare solutions of different mass concentrations (1%, 5%, 10%, and 20%), which were filtered through a 0.22 μm filter and used.

[0114] Soybean oligopeptides and compositions thereof were prepared according to the proportions in Table 1.

[0115] Table 1 Raw material proportions (mass ratio) of soybean oligopeptides and compositions thereof

[0116]

[0117] 2. Experimental methods

[0118] MTT method:

[0119] Human normal liver cells LO2 were cultured in RPMI-1640 medium (containing 10% FBS, 1% PS) at 5% CO2, 37°C constant temperature;

[0120] The logarithmic phase growth of LO2 cells was collected, the cell suspension concentration was adjusted, and the cell suspension was inoculated in a 96-well plate, and the cells were plated to a density of about 10,000 cells per well, 100 μL of cell suspension per well, and incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was removed and washed with PBS. After washing, 100 μL of culture medium containing 1%, 5%, 10%, and 20% of BO1, BO2, and BC1 to BC7 was added to each well, and fresh RPMI-1640 medium (without any soybean oligopeptide) was added as a blank group. The culture plate was placed in a 37°C incubator with 5% CO2. After 24 hours, the culture plate was removed, 10 μl of MTT (5 mg / mL) solution was added to each well, and incubation was continued for 4 hours. The culture was terminated, and the culture solution in the well was carefully aspirated. Then 100 μl of DMSO was added to each well, and the plate was placed on a shaker for 10 minutes to fully dissolve the purple crystals. The absorbance of each well was measured at 490 nm on a full-automatic microplate reader.

[0121] 3. Experimental results

[0122] The experimental results are shown in Table 1. Figure 1

[0123] The results show that, compared with the no-addition group, the addition of different concentrations of BO1, BO2, and BC1 to BC7 has no toxicity to human normal liver cells LO2 and has no significant effect on cell activity.

[0124] Example Three Anti-inflammatory Efficacy Evaluation of Soybean Oligopeptides and Compositions Thereof

[0125] In this experiment, LPS (lipopolysaccharide) was used to induce allergic reactions in RAW264.7 cells to release NO (nitric oxide). The strength of NO inhibition can reflect the size of the anti-inflammatory ability of the sample.

[0126] Experimental method:

[0127] (1) Sample preparation

[0128] A 10 μg / mL LPS PBS solution was prepared as a positive control working solution. Based on the experimental and production costs, 5% and 10% culture medium containing BO1, BO2, or BC1 to BC7 were selected as test samples.

[0129] (2) Cell culture

[0130] RAW264.7 cells were inoculated into a 96-well plate, 100 μl per well, 5×10 3 cells / well.

[0131] Incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0132] ​(3) Sample testing

[0133] After culturing cells for 24 hours, the culture medium was removed and the cells were washed with PBS. After washing, 100 μl of culture medium containing oligopeptides or oligopeptide composition + 1 μl of positive control working solution was added to each well (maintaining a final LPS concentration of 0.1 μg / mL per well). One group was treated as a blank group by adding fresh culture medium without oligopeptides or oligopeptide composition, and the other group was treated as an LPS positive group by adding fresh culture medium without oligopeptides or oligopeptide composition + 1 μl of positive control working solution.

[0134] After adding the sample, the plate was incubated at 37 °C with 5% CO2 for 20 hours. After 20 hours, 50 μl of the culture medium was transferred to a new 96-well plate, and 50 μl of 1% Griessr reagent was added. After mixing, the plate was incubated in the dark for 10 minutes. The absorbance of the sample was measured at 540 nm using a microplate reader, and the results were repeated in triplicate.

[0135] NO release rate % = A / A LPS ×100%

[0136] Among them: A LPS A represents the absorbance of the LPS-positive group sample, and A represents the absorbance of each test sample.

[0137] Experimental results are as follows Figure 2 As shown.

[0138] Higher NO release indicates a more severe inflammatory response. Figure 2 It is known that LPS induces NO production, while BO1 and BO2 have little effect on LPS-induced NO release, and BC1 to BC7 can significantly inhibit LPS-induced NO production, especially BC5. Therefore, the soybean oligopeptide composition with a specific ratio of BC5 exhibits significant anti-inflammatory activity.

[0139] Example 4: Evaluation of the whitening efficacy of soybean oligopeptides and their compositions

[0140] L-tyrosine was dissolved in PBS, with an L-tyrosine content of approximately 0.4 mg / mL. 5% and 10% (w / w) PBS solutions of BO1, BO2, and BC5 were added, respectively. After incubation at 37°C for 10 min, 2 KU units of tyrosinase preheated at 37°C were added to each solution. After reacting for 24 h, the absorbance at 475 nm was measured. PBS was used as a control group.

[0141] Tyrosinase inhibition rate % = (OD of blank group - OD of experimental group) / OD of blank group × 100%.

[0142] Experimental results are as follows Figure 3As shown, compared with the blank group, BC5 can significantly inhibit the activity of tyrosinase, while BO1 and BO2 have no significant inhibitory effect, indicating that the prepared soybean oligopeptide composition has the whitening effect.

[0143] Example Five Evaluation of antioxidant effect of soybean oligopeptide and its composition

[0144] ABTS solution was dissolved in ultrapure water to make the concentration of ABTS reach 7 mM, and potassium persulfate was added to make the concentration of potassium persulfate reach 2.45 mM. Then the solution was placed in the dark at room temperature overnight for 12 to 16 h. The generated ABTS free radical solution was diluted with phosphate buffer (PBS, 0.2 M, pH 7.4) to make the absorbance value at 734 nm reach 0.70. BO1, BO2 and BC5 solutions with different mass concentrations were prepared with PBS. The ABTS solution and the BO1, BO2 and BC5 solutions were mixed in equal volume, shaken for 30 s, reacted in the dark for 10 min, and then the absorbance value of the reaction solution was measured at 734 nm.

[0145] The clearance rate (%) = [1 - (A i -A j ) / A0] x 100%, wherein A0 is the absorbance value of the ABTS solution; A i is the absorbance value of the ABTS solution cleared by the sample solution with different concentrations; and A j is the absorbance value of the sample solution with different concentrations.

[0146] The experimental results are shown in Table 1. Figure 4 As shown, compared with the blank group, BC5 can significantly clear ABTS free radicals, while BO1 and BO2 have no significant clearing effect, indicating that the prepared soybean oligopeptide composition has the antioxidant effect.

[0147] Example Six Proliferation effect of soybean oligopeptide composition on keratinocytes

[0148] In this experiment, the proliferation effect of soybean oligopeptide composition on keratinocytes was evaluated to reflect the repair ability of the sample.

[0149] Experimental method:

[0150] (1) Sample preparation

[0151] BO1, BO2 and BC5 were dissolved in the culture medium respectively and filtered through a 0.22 μm filter to prepare the test solutions with different mass concentrations.

[0152] (2) Cell culture

[0153] Human immortalized keratinocytes (HaCaT) were inoculated into a 96-well plate at 100 μl per well, 5 x 10 3 cells / well.

[0154] Incubate at 37 °C in a 5% CO2 incubator for 24 hours.

[0155] (3) Sample detection

[0156] 1) Drug intervention: after the cells grow completely attached, replace the fresh medium (with or without oligopeptide), 100 μL per well, set 3 replicates, and return to the cell incubator for continuous culture.

[0157] 2) CCK-8 assay: after 24 hours of drug intervention, aspirate the original culture medium, and replace it with a medium containing 10% CCK-8, 100 μL per well. Mix well and continue to incubate for 1 hour.

[0158] 3) Enzyme marker 450 nm wavelength condition for absorbance determination. Cell survival rate (%) = OD value of test sample group / OD value of control group x 100%.

[0159] The experimental results are shown in Table 3. Figure 5 As shown in Table 3, BC5 can effectively promote the proliferation of keratinocytes, and BO1 and BO2 have no significant effect on promoting the proliferation of keratinocytes.

[0160] Preparation of a composition with anti-inflammatory, repair, whitening and antioxidant effects

[0161] According to the formula in Table 2, weigh each raw material

[0162] Table 2: Raw material ratio of each sample (% by mass)

[0163]

[0164] Among them, GMP is a commercially available cosmetic raw material soybean (GLYCINE MAX) polypeptide, and the preservative is selected as phenoxyethanol.

[0165] Each sample is prepared by the following method:

[0166] ① Weigh BO1 and BO2 according to the formula, mix well, prepare BC5, and reserve;

[0167] Or directly according to the formula, weigh BO1, BO2 or GMP;

[0168] ② Weigh 1,3-propanediol, glycerol, preservative and appropriate amount of water according to the formula, mix well, heat to 45 °C under stirring condition, and completely dissolve to obtain phase I;

[0169] ③ Add phase I and appropriate amount of water into the emulsifying pot, start the stirrer and high-speed homogenizer, and homogenize at 50 °C until uniform;

[0170] IV. Close the homogenizer and start to cool down, when the emulsifying kettle is cooled down to room temperature, add BO1, BO2, BC5 or GMP into the emulsifying kettle, and add water to 100%, stir until uniform, to obtain the finished product;

[0171] The finished product is a cosmetic water.

[0172] Example Eight Skin Barrier Repair Efficacy Test

[0173] The test population is 20 volunteers with facial redness, divided into 4 groups of 5 people each, and each group uses samples 1 to 4 in Example Seven of the application.

[0174] Test area: whole face;

[0175] Usage: use twice a day in the morning and evening. Use the German CK company TM300 tester to test the trans-epidermal water loss, TEWL (g / m 2 h), i.e. how many grams of water are lost per square meter per hour. The skin water loss TEWL is a very important parameter for evaluating the function of the skin water protection layer, and has been widely recognized internationally, and can well reflect the integrity of the skin barrier function. The standard TEWL is measured under the condition that the skin is dry and no sweat, at an ambient temperature of 24°C and an ambient humidity of 50%.

[0176] Evaluation criteria: the better the skin protection layer, the higher the water content, and the lower the TEWL value of skin water loss, i.e. the skin barrier is repaired, and the results are shown in Figure 6 .

[0177] From the above data, it can be seen that sample 3 using BC5 as raw material can significantly reduce the trans-epidermal water loss value (TEWL) after a period of use compared with other samples, indicating that the skin barrier repair effect is obvious. Sample 4 prepared using GMP as raw material has a higher TEWL and serious water loss.

[0178] Example Nine Preparation of a Composition with Anti-inflammatory, Repairing, Whitening and Antioxidant Efficacy

[0179] A composition with anti-inflammatory, repairing, whitening and antioxidant efficacy, characterized by comprising the following components in weight percentage:

[0180] Soy oligopeptide composition 8.00 %, 1,3-butanediol 6.00 %, cetyl octanoate 6.00 %, 1,2-hexanediol 7.00 %, glycerin 7.00 %, dimethicone 3.50 %, polyglyceryl-3 methylglucose distearate 1.20 %, cetearyl alcohol 1.50 %, hydroxypropyl starch phosphate 2.00 %, sodium stearoyl glutamate 1.50 %, panthenol 3.00 %, hydrogenated lecithin 1.50 %, tocopheryl acetate 0.10 %, glyceryl octanoate 0.10 %, stearic acid 0.50 %, palmitic acid 0.50 %, disodium EDTA 1.50 %, ethylhexylglycerin 3.50 %, polysorbate-20 1.50 %, sorbitan isostearate 0.50 %, myristic acid 0.10 %, 7-dehydrocholesterol 0.10 %, gluconolactone 1.00 %, lauric acid 1.00 %, tocopherol 0.50 %, preservative 0.05 % and water q.s.;

[0181] The preservative is selected from oxyphenylpropyl ester;

[0182] The composition with anti-inflammatory, repair, whitening and antioxidant efficacy is prepared by the following method:

[0183] ① Soy oligopeptide 1 and soy oligopeptide 2 are weighed and uniformly mixed in a mass ratio of 2:3 to prepare a soy oligopeptide composition for standby;

[0184] ② Cetyl octanoate, 1,2-hexanediol, glycerin, dimethicone, polyglyceryl-3 methylglucose distearate, cetearyl alcohol, hydroxypropyl starch phosphate, panthenol, polysorbate-20 and sorbitan isostearate are weighed according to the formula and uniformly mixed, heated to 70 ℃ under stirring conditions, and completely dissolved to obtain phase I;

[0185] ③ Phase I and appropriate amount of water are added to the emulsifying kettle, the stirrer and high-speed homogenizer are started, and homogenization is carried out at 65 ℃ until uniform;

[0186] ④ The heating is turned off and the temperature is started to be reduced, when the emulsifying kettle is cooled to 45 ℃, sodium stearoyl glutamate, hydrogenated lecithin, tocopheryl acetate, glyceryl octanoate, stearic acid, palmitic acid, disodium EDTA, myristic acid, 7-dehydrocholesterol, gluconolactone, lauric acid, tocopherol, preservative and appropriate amount of water are added to the emulsifying kettle, and homogenized for 5 min, when the temperature of the emulsifying kettle is reduced to 35 ℃, ethylhexylglycerin is added to the emulsifying kettle, and homogenized for 5 min;

[0187] ⑤ When the emulsifying kettle is cooled to room temperature, the soy oligopeptide composition is added to the emulsifying kettle, and the water is supplemented to 100 %, and stirred until uniform to obtain the finished product;

[0188] The finished product is a cream.

[0189] The above description is further detailed in connection with specific preferred embodiments of the present application, and it is not to be construed that the specific implementation of the present application is limited to these descriptions. For those skilled in the art to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can be made, and all of them should be considered as falling within the protection scope of the present application.

Claims

1. A soy oligopeptide composition, characterized in that, consisting of soy oligopeptide 1 and soy oligopeptide 2; the amino acid sequence of the soy oligopeptide 1 is SEQ ID No. 1; the amino acid sequence of the soy oligopeptide 2 is SEQ ID No.

2.

2. The soy oligopeptide composition according to claim 1, characterized in that, The mass ratio of the soy oligopeptide 1 and the soy oligopeptide 2 is (1-3):(3-1).

3. The soy oligopeptide composition according to claim 1 or 2, characterized in that, The mass ratio of the soy oligopeptide 1 and the soy oligopeptide 2 is 2:

3.

4. A composition having anti-inflammatory, reparative, whitening and antioxidant efficacy, characterized in that, comprising the following components in the following mass percentage: The soy oligopeptide composition according to any one of claims 1-3 10.00 %, 1,3-propanediol 30.00 %, glycerin 45.00 %, preservative 0.05 % and water balance; The preservative is selected from one or more of methylparaben, propylparaben, phenoxyethanol, chlorphenesin, benzisidene alkylthiazolinone.

5. A composition having anti-inflammatory, reparative, whitening and antioxidant efficacy, characterized in that, comprising the following components in the following mass percentage: The soy oligopeptide composition according to any one of claims 1-3 8.00 %, 1,3-butanediol 6.00 %, cetyl octanoate 6.00 %, 1,2-hexanediol 7.00 %, glycerin 7.00 %, dimethicone 3.50 %, polyglyceryl-3 methylglucose distearate 1.20 %, cetyl stearyl alcohol 1.50 %, hydroxypropyl starch phosphate 2.00 %, sodium stearoyl glutamate 1.50 %, panthenol 3.00 %, hydrogenated lecithin 1.50 %, tocopheryl acetate 0.10 %, glyceryl caprylate 0.10 %, stearic acid 0.50 %, palmitic acid 0.50 %, disodium EDTA 1.50 %, ethylhexylglycerin 3.50 %, polysorbate-20 1.50 %, sorbitan isostearate 0.50 %, myristic acid 0.10 %, 7-dehydrocholesterol 0.10 %, glycolate lactone 1.00 %, lauric acid 1.00 %, tocopherol 0.50 %, preservative 0.05 % and water balance; The preservative is selected from one or more of methylparaben, propylparaben, phenoxyethanol, chlorphenesin, benzisidene alkylthiazolinone.

6. Use of the soy oligopeptide composition according to any one of claims 1-3 in the preparation of a cosmetic product having anti-inflammatory, repairing, whitening and antioxidant efficacy. ​

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