Oil body protein, and preparation method and application thereof

Abstract

The present application relates to the technical field of cosmetic functional materials, in particular to an oil body protein, a preparation method and application thereof.The amino acid sequence of the oil body protein is shown in SEQ ID No.1 and / or SEQ ID No.2.The oil body protein has the advantages of simple structure and simple synthesis method, can effectively promote the expression of collagen, is more suitable for actual production needs, and can be applied to the preparation of cosmetics and / or medical products.

Description

Technical Field

[0001] This invention relates to the field of cosmetic efficacy raw material technology, and more specifically, to an oil-based protein, its preparation method, and its application. Background Technology

[0002] Oil body membrane proteins (oil body proteins) are inherent components on the surface of plant oil bodies and are usually found in the seeds of various plants. Oil body membrane proteins play a key role in maintaining seed germination, influencing the size of oil bodies in seeds, and lipid accumulation during plant growth and development.

[0003] There are many types of plant oil body membrane proteins. To study the efficacy of a single oil body membrane protein, further purification is required. At present, the separation and purification method is to separate them using protein electrophoresis, which cannot achieve mass production and industrial application, thus limiting their further application.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an oil body protein, its preparation method, and its application. The oil body protein has a simple structure and can effectively promote collagen expression.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0007] One aspect of the present invention relates to an oil body protein having an amino acid sequence as shown in SEQ ID No. 1 and / or SEQ ID No. 2.

[0008] The oil body protein has advantages such as simple structure and simple synthesis method, which can effectively promote collagen expression and is more suitable for actual production needs. It can be used to prepare cosmetics and / or pharmaceuticals.

[0009] Another aspect of the present invention relates to a method for preparing the aforementioned oil body protein, comprising the following steps:

[0010] (a) Piperidine and N,N-dimethylformamide were added to the carrier resin, followed by a first reaction and a first wash;

[0011] (b) Add the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine to the first washed reaction solution and carry out the first stirring reaction;

[0012] (c) The carrier resin after the first stirring reaction is subjected to ninhydrin colorimetric detection. If the detection result is negative, the reaction solution of the first stirring reaction is removed by vacuum and N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine are added to carry out a second stirring reaction. The reaction solution of the second stirring reaction is removed by vacuum and a second washing is carried out.

[0013] (d) Add piperidine and N,N-dimethylformamide to the reaction system after the second washing, and then carry out the first deprotection reaction and the third washing;

[0014] (e) Add the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide to the reaction system after the third washing, and then carry out the third stirring reaction and the fourth washing; perform ninhydrin colorimetric detection on the carrier resin, and if the detection result is negative, add piperidine and N,N-dimethylformamide to the reaction system after the fourth washing to carry out the second deprotection reaction and the fifth washing;

[0015] (f) According to the amino acid sequence of the oil body protein, the second amino acid raw material is replaced sequentially with the third amino acid raw material and the amino acid raw material after the third position, and step (e) is repeated to synthesize the amino acid sequence of the oil body protein.

[0016] (g) The reaction system after step (f) is subjected to end-capping reaction and sixth washing; the carrier resin is subjected to ninhydrin colorimetric detection. If the detection result is negative, the carrier resin is subjected to shrinkage washing and first drying to obtain peptide resin; the peptide resin is cleaved and filtered, the filtrate is mixed with methyl tert-butyl ether and then subjected to first centrifugation. After removing the supernatant, crude peptide is obtained; the crude peptide is mixed with methyl tert-butyl ether and then subjected to second centrifugation. After removing the supernatant, it is subjected to second drying to obtain crude product;

[0017] (h) The crude product is purified and converted to salt.

[0018] The method for preparing the oil body protein is simple, suitable for industrial mass production, and the prepared oil body protein has a simple structure, high purity, and excellent effect on promoting collagen expression. It can be effectively applied in cosmetics and / or pharmaceuticals.

[0019] Another aspect of the present invention relates to the use of the oil body protein or the oil body protein prepared by the method of preparing the oil body protein in the preparation of cosmetics and / or pharmaceutical products.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The oil body protein provided by the present invention has the advantages of simple structure and simple synthesis method, which can effectively promote collagen expression and is also more suitable for actual production needs. It can be applied to the preparation of cosmetics and / or pharmaceutical products.

[0022] (2) The method for preparing oil body protein provided by the present invention effectively solves the problem that it is difficult to obtain high-purity natural oil body protein in the existing process. The process is simple and suitable for industrial mass production. The prepared oil body protein has a simple structure, high purity, and excellent effect on promoting collagen expression. It can be effectively applied in cosmetics and / or pharmaceuticals. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 The HPLC chromatogram of the oil body protein SPVIVP;

[0025] Figure 2 This is the HPLC chromatogram of the oil body protein SPALVP. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0027] One aspect of the present invention relates to an oil body protein having an amino acid sequence as shown in SEQ ID No. 1 and / or SEQ ID No. 2.

[0028] The oil body protein described above has advantages such as simple structure and simple synthesis method. It can effectively promote collagen expression and is more suitable for actual production needs. It can be used to prepare cosmetics and / or pharmaceutical products.

[0029] Furthermore, the amino acid sequence of the oil body protein is Serine-Proline-Valine-Isoleucine-Valine-Proline and / or Serine-Proline-Valine-Leucine-Valine-Proline.

[0030] Furthermore, SEQ ID No. 1: Ser-Pro-Val-Iso-Val-Pro (abbreviated as SPVIVP).

[0031] Furthermore, SEQ ID No. 2: Ser-Pro-Val-Leu-Val-Pro (abbreviated as SPVLVP).

[0032] Another aspect of the present invention relates to a method for preparing the aforementioned oil body protein, comprising the following steps:

[0033] (a) Piperidine and N,N-dimethylformamide were added to the carrier resin, followed by a first reaction and a first wash;

[0034] (b) Add the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine to the first washed reaction solution and carry out the first stirring reaction;

[0035] (c) The carrier resin after the first stirring reaction is subjected to ninhydrin colorimetric detection. If the detection result is negative, the reaction solution of the first stirring reaction is removed by vacuum and N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine are added to carry out a second stirring reaction. The reaction solution of the second stirring reaction is removed by vacuum and a second washing is carried out.

[0036] (d) Add piperidine and N,N-dimethylformamide to the reaction system after the second washing, and then carry out the first deprotection reaction and the third washing;

[0037] (e) Add the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide to the reaction system after the third washing, and then carry out the third stirring reaction and the fourth washing; perform ninhydrin colorimetric detection on the carrier resin, and if the detection result is negative, add piperidine and N,N-dimethylformamide to the reaction system after the fourth washing to carry out the second deprotection reaction and the fifth washing;

[0038] (f) According to the amino acid sequence of the oil body protein, the second amino acid raw material is replaced sequentially with the third amino acid raw material and the amino acid raw material after the third position, and step (e) is repeated to synthesize the amino acid sequence of the oil body protein.

[0039] (g) The reaction system after step (f) is subjected to end-capping reaction and sixth washing; the carrier resin is subjected to ninhydrin colorimetric detection. If the detection result is negative, the carrier resin is subjected to shrinkage washing and first drying to obtain peptide resin; the peptide resin is cleaved and filtered, the filtrate is mixed with methyl tert-butyl ether and then subjected to first centrifugation. After removing the supernatant, crude peptide is obtained; the crude peptide is mixed with methyl tert-butyl ether and then subjected to second centrifugation. After removing the supernatant, it is subjected to second drying to obtain crude product;

[0040] (h) The crude product is purified and converted to salt.

[0041] The method for preparing the described oil body protein effectively solves the problem of obtaining high-purity natural oil body protein in existing processes. The process is simple, suitable for industrial mass production, and the prepared oil body protein has a simple structure, high purity, and excellent effect on promoting collagen expression. It can be effectively applied in cosmetics and / or pharmaceuticals.

[0042] The first amino acid raw material mentioned in this invention refers to the amino acid raw material required for synthesizing the first amino acid of oil body protein, and the subsequent amino acid raw materials are derived sequentially. For example, if the first amino acid of oil body protein is proline, the first amino acid raw material is Fmoc-Pro-OH; if the second amino acid is valine, the second amino acid raw material is Fmoc-Val-OH.

[0043] This invention does not specifically limit the type of carrier resin; any conventional carrier resin in the art can be used to implement the technical solutions of this invention. In some specific embodiments, the carrier resin includes, but is not limited to, Rink Amide-AM resin.

[0044] Further, in step (a), the ratio of the carrier resin, piperidine, and N,N-dimethylformamide added is 50g:60mL:240mL. Adding the carrier resin, piperidine, and N,N-dimethylformamide in a specific amount ensures sufficient reaction between the carrier resin and the piperidine and N,N-dimethylformamide.

[0045] Furthermore, the first reaction time is 25 minutes. By limiting the first reaction time within a certain range, the reactants can react completely.

[0046] Furthermore, the first wash was performed 7 times, each time using 500 mL of washing solution.

[0047] Further, in step (b), the addition ratio of the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-dimethylformamide, and N,N-diisopropylethylamine is 100 mmol: 40.7 g: 210 mL: 35 mL. Limiting the amount of materials added in step (b) ensures that the first stirring reaction proceeds fully.

[0048] Furthermore, the first stirring reaction takes 2 hours.

[0049] Furthermore, the carrier resin was subjected to ninhydrin colorimetric detection. If the carrier resin did not show color, the test result was negative.

[0050] Further, in step (c), the addition ratio of N,N-dimethylformamide, acetic anhydride, and N,N-diisopropylethylamine is 280 mL: 10 mL: 10 mL. Limiting the amount of raw materials added in step (c) ensures that the second stirring reaction proceeds sufficiently.

[0051] Furthermore, the second stirring reaction time is 0.5 h.

[0052] Furthermore, the second washing is repeated 3 times, with the volume ratio of the washing liquid to the carrier resin being 1:10 each time.

[0053] Further, in step (d), piperidine and N,N-dimethylformamide are added to the reaction system after the second washing in an amount of 120 mL piperidine and 480 mL N,N-dimethylformamide. Limiting the amount of piperidine and N,N-dimethylformamide added ensures that the first deprotection reaction proceeds sufficiently.

[0054] Furthermore, the first deprotection reaction takes 25 minutes.

[0055] Furthermore, the third washing is repeated 7 times, with the volume ratio of the washing liquid to the carrier resin being 1:10 each time.

[0056] Further, in step (e), the addition ratio of the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide, and N,N-dimethylformamide is 100 mmol: 100 mmol: 100 mmol: 280 mL. Limiting the amount of reactants added for the third stirring reaction ensures that the reaction proceeds fully and avoids the formation of byproducts.

[0057] Furthermore, the third stirring reaction takes place for 1.5 hours.

[0058] Furthermore, the fourth washing is repeated 3 times, with the volume ratio of the washing liquid to the carrier resin being 1:10 each time.

[0059] Further, in step (e), piperidine and N,N-dimethylformamide are added to the reaction system after the fourth washing in an amount of 120 mL and 480 mL, respectively. Limiting the amount of piperidine and N,N-dimethylformamide added during the second deprotection reaction ensures that the second deprotection reaction is complete.

[0060] Furthermore, the second deprotection reaction takes 25 minutes.

[0061] Furthermore, the fifth washing is repeated 5 times, with the volume ratio of the washing liquid to the carrier resin being 1:10 each time.

[0062] Further, 320 mL of capping solution was added to the reaction system after step (f) to carry out the capping reaction.

[0063] Furthermore, the capping solution for the capping reaction comprises N,N-dimethylformamide, N,N-diisopropylethylamine (DIEA), and acetic anhydride (Ace) in a volume ratio of 28:1:1.

[0064] Furthermore, the capping reaction time is 0.5 h.

[0065] Furthermore, the sixth washing is repeated 3 times, with the volume ratio of the washing liquid to the carrier resin being 1:10 each time.

[0066] Furthermore, the shrinkage wash uses 350 mL of methanol as the washing solution.

[0067] Furthermore, the pyrolysis solution comprises trifluoroacetic acid (TFA), 1,2-ethylenedithiol (EDT), and water in a volume ratio of 90:5:5.

[0068] Furthermore, the ratio of peptide resin to lysis buffer is 1g:8mL. Maintaining this ratio within a reasonable range ensures thorough lysis of the peptide resin.

[0069] Furthermore, the pyrolysis time is 3 hours.

[0070] Furthermore, during the first centrifugation, the volume of methoxymethyl tert-butyl ether added is 8 times the volume of the filtrate.

[0071] Furthermore, during the second centrifugation, the volume of methoxymethyl tert-butyl ether added is 8 times the volume of the filtrate.

[0072] Furthermore, the first centrifugation speed is 3000 r / min, and the first centrifugation time is 2 min.

[0073] Furthermore, the second centrifugation speed is 3000 r / min, and the second centrifugation time is 2 min.

[0074] Further purification was performed using liquid chromatography.

[0075] Furthermore, the purified mobile phase A is a 0.1 vol% aqueous acetic acid solution, the mobile phase B is a 0.1 vol% acetic acid acetonitrile solution, and the detection wavelength is 220 nm or 254 nm.

[0076] Furthermore, the salt transfer was carried out using a 0.3 vol% aqueous acetic acid solution.

[0077] Furthermore, the washing solutions for the first wash, the second wash, the third wash, the fourth wash, the fifth wash, and the sixth wash include, but are not limited to, N,N-dimethylformamide.

[0078] Another aspect of the present invention relates to the use of the oil body protein or the oil body protein prepared by the method of preparing the oil body protein in the preparation of cosmetics and / or pharmaceutical products.

[0079] The raw material information for preparing oil body protein in the specific embodiments of this application is shown in Table 1.

[0080] Table 1

[0081]

[0082] Information on reagents for preparing oil body protein in the specific embodiments of this application is shown in Table 2.

[0083] Table 2

[0084]

[0085] Information on the preparation of resins for oil-body proteins in the specific embodiments of this application is shown in Table 3.

[0086] Table 3

[0087]

[0088] This invention uses HPLC to test purity and content.

[0089] This invention uses HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry) for component identification.

[0090] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0091] Example 1

[0092] The method for preparing an oil body protein with the amino acid sequence SPVIVP provided in this embodiment includes the following steps:

[0093] a. Weigh 50g of Rink Amide-AM resin and place it in a polypeptide reactor. Add 60mL of piperidine and 240mL of industrial-grade N,N-dimethylformamide and react for 25min. Then add DMF and wash 7 times, each time using 500mL.

[0094] b. Weigh 100 mmol of amino acids (Fmoc-Pro-OH) and 40.7 g of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, add 210 mL of AR grade N,N-dimethylformamide and 35 mL of N,N-diisopropylethylamine, stir well and add to the peptide reactor, turn on the peptide reactor and stir for 2 h;

[0095] c. The resin was tested for ninhydrin and the result was negative, indicating that the degree of substitution of Rink Amide-AM resin was 0.93 mmol / g;

[0096] d. After vacuuming away the above reaction solution, add 280 mL of AR grade N,N-dimethylformamide, 10 mL of acetic anhydride, and 10 mL of N,N-diisopropylethylamine and stir for 0.5 h. Vacuum dry and wash three times with industrial grade N,N-dimethylformamide, each time using 10 times the volume of resin.

[0097] e. Add 600 mL of a 20% piperidine / N,N-dimethylformamide solution (i.e., 120 mL piperidine and 480 mL N,N-dimethylformamide) to deprotect the resin for 25 min. After the reaction, wash the resin 7 times with industrial-grade N,N-dimethylformamide, with each wash using approximately 10 times the volume of the resin.

[0098] f. Condensation was performed using a system of ethyl 2-oxime cyanoacetate and N,N'-diisopropylcarbodiimide (Oxyma, DIC). The feed amounts of Fmoc-Val-OH, ethyl 2-oxime cyanoacetate, and N,N'-diisopropylcarbodiimide were all 100 mmol. 280 mL of AR-grade N,N-dimethylformamide was added, and the mixture was stirred for 1.5 h. After condensation, industrial-grade N,N-dimethylformamide was added for washing three times, with each wash volume being 10 times the resin volume. The ninhydrin test on the carrier resin was negative. 600 mL of a 20% piperidine / N,N-dimethylformamide solution (i.e., 120 mL piperidine and 480 mL N,N-dimethylformamide) was added for deprotection for 25 min. After deprotection, industrial-grade N,N-dimethylformamide was added for washing five times, with each wash volume being 10 times the resin volume.

[0099] g. Following the amino acid sequence (Pro-Val-Ile-Val-Pro-Ser), repeat the condensation reaction-washing-detection-deprotection-washing process in step (f) until the sequence condensation is complete;

[0100] h. Capping solution preparation: N,N-dimethylformamide: DIEA: Ace = 28:1:1 (v / v / v), add 320 mL of capping solution, capping reaction for 0.5 h, after the reaction is completed, wash 3 times with N,N-dimethylformamide, each wash volume is about 10 times the resin volume; ninhydrin test of the carrier resin is negative; add 350 mL of methanol to shrink and wash the carrier resin, then vacuum dry the resin for 5 h to obtain 77.5 g peptide resin;

[0101] i. Preparation of lysis buffer: trifluoroacetic acid: 1,2-ethylenedithiol: water = 90:5:5 (v / v / v);

[0102] j. Add lysis buffer at a ratio of 1 g: 8 mL for peptide resin, stir at room temperature for 3 h, then filter (to remove resin). Add 8 times the volume of methacin to the filtrate, stir for 5 min, then centrifuge at 3000 r / min for 2 min. Remove the supernatant to obtain crude peptide. Add the same volume of methacin as the first time, disperse evenly, centrifuge again to obtain a white solid, freeze-dry under vacuum for 8 h to obtain crude product. Samples were sent for MS and HPLC analysis and confirmed as the target analyte.

[0103] k. The crude product was purified using preparative liquid chromatography (HPLC). Mobile phase: A: 0.1 vol% acetic acid aqueous solution; B: 0.1 vol% acetic acid acetonitrile solution. Detection wavelength: λ = 220 nm. Purity ≥ 95%. The target analyte was collected. The purified peptide was then transsalted using 0.3 vol% acetic acid aqueous solution. The transsalted sample was collected, with a purity ≥ 95%. Figure 1 The yield of pure polypeptide was 66.3%.

[0104] l. Place the sample solution obtained after salt conversion into a freeze dryer and freeze dry at <20 Pa for 76 h to obtain a white powder; dissolve the obtained white powder in a 5 vol% acetonitrile aqueous solution and then freeze dry it again for 65-80 h to obtain the final product.

[0105] Example 2

[0106] The method for preparing oleosome protein with the amino acid sequence of SPLVVP provided in this embodiment includes the following steps:

[0107] a~f. Same as Example 1;

[0108] g. Following the amino acid sequence (Pro-Val-Leu-Val-Pro-Ser), repeat the condensation reaction-washing-detection-deprotection-washing process in step (f) until the sequence condensation is complete;

[0109] h~j. Same as Example 1;

[0110] k. The crude product was purified using preparative liquid chromatography (HPLC). Mobile phase: A: 0.1 vol% acetic acid aqueous solution; B: 0.1 vol% acetic acid acetonitrile solution. Detection wavelength: λ = 220 nm. Purity ≥ 95%. The target analyte was collected. The purified peptide was then transsalted using 0.3 vol% acetic acid aqueous solution. The transsalted sample was collected, with a purity ≥ 95%. Figure 2 The yield of pure polypeptide was 62.9%.

[0111] l. Same as Example 1.

[0112] Experimental Example 1

[0113] Assay to promote collagen-I expression: A method for detecting COL-I expression in fibroblasts.

[0114] (1) Inoculation: Inoculate cells into 24-well plates and incubate at 37°C in a 5% CO2 incubator for 18-24 hours.

[0115] (2) Solution preparation: Prepare test and control samples according to Table 4. The sample group is prepared by first dissolving the oil body protein in DMSO (the mass ratio of oil body protein to DMSO is 1:10), and then adding it to the cell culture medium to dilute to the corresponding mass concentration. The negative control group is added to the cell culture medium containing 0.1% DMSO.

[0116] (3) Sample feeding: According to the experimental grouping and concentration settings in Table 5, after the cells in the 24-well plate have grown for 18-24 hours, samples are fed in groups, with 3 replicates per group. The cells are then cultured at 37°C in a 5% CO2 incubator for 48 hours.

[0117] (4) Detection of type I collagen content: Take the supernatant and use an ELISA kit for detection.

[0118] (5) Data processing: All data obtained in the experiment were processed and plotted using Excel software. SPSS 17.0 was used for statistical analysis. One-way ANOVA was used for comparisons between groups. A p < 0.05 was considered statistically significant.

[0119] The test groups are shown in Table 4.

[0120] Table 4. Efficacy Test Groups and Test Concentrations (col-I)

[0121]

[0122] Table 5. Results of col-I content test data

[0123]

[0124] The test results show that the addition of liposome proteins SPVIVP and SPPVLVP at concentrations of 0.0005% to 0.01% can effectively promote the increase of type I collagen content in fibroblasts, and the promoting effect is positively correlated with the content of liposome protein fragments; and there is a statistically significant difference compared with the control group.

[0125] Experiment Example 2

[0126] Matrix metalloproteinase-1 assay, based on the detection method of MMP-1 expression in fibroblasts:

[0127] (1) Inoculation: Inoculate cells into 24-well plates and incubate at 37°C in a 5% CO2 incubator for 18-24 hours.

[0128] (2) Solution preparation: Prepare the test substance and positive control according to Table 6. The sample group is prepared by first dissolving the oil body protein in DMSO (the mass ratio of oil body protein to DMSO is 1:10), and then adding it to the cell culture medium to dilute to the corresponding mass concentration. The negative control group is prepared by adding cell culture medium containing 0.1% DMSO.

[0129] (3) Sample feeding: According to the experimental grouping and concentration settings in Table 7, after the cells in the 24-well plate have grown for 18-24 hours, samples are fed in groups, with 3 replicates per group. The cells are then cultured at 37°C in a 5% CO2 incubator for 48 hours.

[0130] (4) Detection of matrix metalloproteinase-1 content: Take the supernatant and detect it using an ELISA kit.

[0131] (5) Data processing: All data obtained in the experiment were processed and plotted using Excel software. Statistical analysis was performed using SPSS 17.0. One-way ANOVA was used for comparisons between groups, and a p-value < 0.05 was considered statistically significant.

[0132] Table 6. Efficacy Test Groups and Test Concentrations (MMP-1)

[0133]

[0134] Table 7. Results of MMP-1 content test.

[0135]

[0136] Note: The addition of 0.01% VA alcohol will reduce fibroblast viability to below 50%, therefore it will not be investigated.

[0137] The test results show that the oil body protein fragments SPVIVP and SPPVLVP, at an addition level of 0.0005%~0.01%, can effectively inhibit the activity of type I matrix metalloproteinases in fibroblasts, prevent excessive MMP-1 activity in the skin from damaging the integrity of the skin structure, and reduce the rate of collagen decomposition; and compared with the control group, there is a statistically significant difference.

[0138] Experimental Example 3

[0139] The collagen-XVII assay, a keratinocyte-based detection method:

[0140] (1) Inoculation: Inoculate cells into 24-well plates and incubate at 37°C in a 5% CO2 incubator for 18-24 hours.

[0141] (2) Solution preparation: Prepare the test substance and positive control according to Table 8. The sample group is prepared by first dissolving the oil body protein in DMSO (the mass ratio of oil body protein to DMSO is 1:10), and then adding it to the cell culture medium to dilute to the corresponding mass concentration.

[0142] (3) Sample delivery: According to the experimental grouping and concentration settings in Table 9, after the cells in the 24-well plates have grown for 18-24 hours, samples are delivered in groups, with 3 replicates per group. The blank control and negative control groups are given cell culture medium containing 0.1% DMSO, the sample groups are given complete cell culture medium containing the corresponding concentration of the sample, the positive control is given cell culture medium containing 10 ng / mL hEGF, and the sample groups are given complete cell culture medium containing the corresponding concentration of the sample. The cells are then incubated at 37°C in a 5% CO2 incubator for 18-24 hours.

[0143] (4) COL-17 expression detection: Immunofluorescence staining was performed, followed by washing three times with PBS, fixation with 4% paraformaldehyde for 30 min, washing three times with PBS, permeabilization with 0.5% Triton X-100 for 20 min, washing three times with PBS, blocking with 5% BSA at room temperature for 60 min, and incubation with primary antibody at 4℃ overnight. The next day, the cells were washed three times with PBS, incubated with secondary antibody at room temperature for 1 h, and then washed three times with PBS. The cells were observed and photographed under a fluorescence microscope.

[0144] (5) Data processing: ImageJ software was used to process fluorescence photographs and calculate IOD values. The obtained data were processed and plotted using Excel software. SPSS 17.0 was used for statistical analysis, and one-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0145] Table 8. Efficacy Test Groups and Test Concentrations (col-XVII)

[0146]

[0147] Table 9 Results of col-XVII content test.

[0148]

[0149] The test results show that the oil body protein fragments SPVIVP and SPPVLVP can effectively promote the increase of type XVII collagen content in fibroblasts. At an addition of 0.001%, the promoting effect is 15.5 times that of the control group. The promoting effect is positively correlated with the content of oil body protein fragments, which indicates that the invention has the function of supporting the structural stability of the skin. Moreover, it has a statistically significant difference compared with the blank group.

[0150] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An oil-body protein, characterized in that, The amino acid sequence of the oil body protein is shown in SEQ ID No. 1 or SEQ ID No.

2.

2. The method for preparing oil-body protein as described in claim 1, characterized in that, Includes the following steps: (a) Piperidine and N,N-dimethylformamide were added to the carrier resin, followed by a first reaction and a first wash; (b) Add the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine to the first washed reaction solution and carry out the first stirring reaction; (c) The carrier resin after the first stirring reaction is subjected to ninhydrin colorimetric detection. If the detection result is negative, the reaction solution of the first stirring reaction is removed by vacuum and N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine are added to carry out a second stirring reaction. The reaction solution of the second stirring reaction is removed by vacuum and a second washing is carried out. (d) Add piperidine and N,N-dimethylformamide to the reaction system after the second washing, and then carry out the first deprotection reaction and the third washing; (e) Add the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide to the reaction system after the third washing, and then carry out the third stirring reaction and the fourth washing; perform ninhydrin colorimetric detection on the carrier resin, and if the detection result is negative, add piperidine and N,N-dimethylformamide to the reaction system after the fourth washing to carry out the second deprotection reaction and the fifth washing; (f) According to the amino acid sequence of the oil body protein, the second amino acid raw material is replaced sequentially with the third amino acid raw material and the amino acid raw material after the third position, and step (e) is repeated to synthesize the amino acid sequence of the oil body protein. (g) The reaction system after step (f) is subjected to end-capping reaction and sixth washing; the carrier resin is subjected to ninhydrin colorimetric detection. When the detection result is negative, the carrier resin is subjected to shrinkage washing and first drying to obtain peptide resin; the peptide resin is cleaved and filtered, and the filtrate is mixed with methacin and centrifuged for the first time. After removing the upper liquid, crude peptide is obtained. The crude polypeptide and meth tert-butyl ether were mixed and then centrifuged a second time. After removing the supernatant, the mixture was dried a second time to obtain the crude product. (h) The crude product is purified and converted to salt.

3. The method for preparing oil-body protein according to claim 2, characterized in that, Includes at least one of the following technical features: (1) In step (a), the ratio of the carrier resin, piperidine and N,N-dimethylformamide is 50g:60mL:240mL; (2) The reaction time for the first reaction is 25 min; (3) The first wash is performed 7 times, each time using 500 mL of washing solution.

4. The method for preparing oil-body protein according to claim 2, characterized in that, Includes at least one of the following technical features: (1) In step (b), the addition ratio of the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine is 100mmol:40.7g:210mL:35mL; (2) The first stirring reaction time is 2 hours.

5. The method for preparing oil-body protein according to claim 2, characterized in that, Includes at least one of the following technical features: (1) In step (c), the addition ratio of N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine is 280mL:10mL:10mL; (2) The second stirring reaction time is 0.5 h; (3) The second washing is repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:

10.

6. The method for preparing oil-body protein according to claim 2, characterized in that, Includes at least one of the following technical features: (1) Step (d): Piperidine and N,N-dimethylformamide are added to the reaction system after the second washing in an amount of 120 mL piperidine and 480 mL N,N-dimethylformamide. (2) The time for the first deprotection reaction is 25 min; (3) The third washing is repeated 7 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:

10.

7. The method for preparing oil-body protein according to claim 2, characterized in that, Includes at least one of the following technical features: (1) In step (e), the addition ratio of the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide is 100 mmol: 100 mmol: 100 mmol: 280 mL; (2) The third stirring reaction time is 1.5 h; (3) The fourth washing is repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:10; (4) Step (e): Piperidine and N,N-dimethylformamide are added to the reaction system after the fourth washing in an amount of 120 mL piperidine and 480 mL N,N-dimethylformamide. (5) The second deprotection reaction takes 25 minutes; (6) The fifth washing is repeated 5 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:

10.

8. The method for preparing oil-body protein according to claim 2, characterized in that, Includes at least one of the following technical features: (1) After step (f) is completed, add 320 mL of end-capping solution to the reaction system and carry out the end-capping reaction; (2) The capping solution of the capping reaction includes N,N-dimethylformamide, N,N-diisopropylethylamine and acetic anhydride in a volume ratio of 28:1:1; (3) The end-capping reaction time is 0.5 h; (4) The sixth washing is repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:

10.

9. The method for preparing oil-body protein according to claim 2, characterized in that, Includes at least one of the following technical features: (1) The shrinkage washing uses 350 mL of methanol as the washing solution; (2) The pyrolysis solution comprises trifluoroacetic acid, 1,2-ethylenedithiol and water in a volume ratio of 90:5:5; (3) The ratio of the peptide resin to the lysis buffer is 1g:8mL; (4) The pyrolysis time is 3 hours.

10. The use of the oil body protein as described in claim 1 or the oil body protein prepared by the preparation method of any one of claims 2 to 9 in the preparation of cosmetics.

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