Composition containing leukocyte extract and white tomato extract and application thereof in cosmetics

Abstract

The present application relates to a kind of compositions containing leukocyte extract and white tomato extract and its application in cosmetics, belong to the field of cosmetics and biotechnology. Specifically, the present application selects animal umbilical cord blood as raw material, obtains leukocyte extract by extraction, purification step;At the same time, white tomato polypeptide is extracted, and further separation obtains the polypeptide described in SEQ ID NO.1-2;The leukocyte extract and white tomato polypeptide have good safety, and the both synergies have significant antioxidant and whitening effect, can be safely used in the field of cosmetics, expands the application prospect of leukocyte extract and white tomato polypeptide.

Description

Technical Field

[0001] The present invention relates to the fields of cosmetics and biotechnology, and in particular to a composition containing a leukocyte extract and a white tomato extract and application thereof in cosmetics. Background Art

[0002] Leukocytes are classified as neutrophils, eosinophils, basophils, monocytes, and lymphocytes. They are a very important type of cell in the blood, capable of phagocytizing foreign matter and producing antibodies, healing body injuries, and resisting pathogen invasion. Leukocyte extracts contain a large amount of cell-specific proteins, lipids, and nucleic acids, which can act directly on the mucous membranes. They have high transdermal absorption rates and promote proliferation and division of fibroblasts, epithelial cells, endothelial cells, and other cells, as well as angiogenesis, mucosal repair, and cell regeneration. These components can effectively activate senescent cells, promote cell regeneration, repair inflammatory damage, enhance skin immunity, reduce pigmentation, and other effects, helping cells in the body restore normal function and vitality. Therefore, leukocyte extracts are often used as antioxidant drugs or added to skin care products. Although leukocyte extracts can have excellent biological activity, they do not effectively promote skin cell proliferation and metabolism. In other words, the direct use of leukocyte extracts, which are mainly composed of nucleotides, in beauty and skin care products has not been ideal. There is a small amount of growth factors in the blood. After the white blood cells are extracted using traditional extraction methods, the growth factors present in the extract are negligible.

[0003] White tomatoes (Solanum lycopersicum) are annual or perennial herbs in the Solanaceae family, genus Solanum. They grow 2.5 to 4 meters tall or indeterminately. Their branches are covered with sticky glandular hairs and have a strong odor. Their stems are prone to lodging. Their leaves are pinnate or pinnately lobed, and their inflorescences have a peduncle length of 10 to 15 centimeters, often with 6 to 12 flowers. These indeterminate tomatoes weigh 150 to 200 grams each. When ripe, they turn golden yellow, creating a vibrant appearance, high quality, and firm, long-lasting storage. White tomatoes not only contain the carotene, vitamin C, and B vitamins found in regular tomatoes, but are also rich in colorless carotene, phytoene, phytoene, and phytoene, which have a unique whitening effect. These ingredients are absent or found in low concentrations in other colored tomatoes. In addition, the existing technology rarely discloses the extraction and application of white tomato peptides, which can supplement the collagen needed by the human body. Collagen is an important component of cartilage and connective tissue, and also helps maintain skin elasticity and moisture balance. At the same time, it helps improve the body's resistance and play a role in enhancing immunity.

[0004] CN114191373A discloses a rapid and simple method for extracting a leukocyte extract from umbilical cord blood, comprising the following steps: S1, collecting human umbilical cord blood; S2, centrifuging the umbilical cord blood obtained in step S1 to obtain the buffy coat supernatant, i.e., umbilical cord blood plasma containing the leukocyte layer, and freezing and preserving the plasma; S3, thawing the plasma frozen in step S2 and performing infectious disease antibody testing, viral RT-PCR fluorescence or PCR fluorescence quantitative testing; S4, testing for endotoxins in the plasma; S5, centrifuging the plasma that passes both steps S3 and S4, filtering it after centrifugation, and finally freezing and preserving it. This method requires the collection of human leukocyte extracts from human blood, and the yield is relatively low.

[0005] CN119606831A discloses a white tomato whitening and repairing composition, its preparation method, and its application. The composition comprises, by mass percentage, 68-85% jojoba oil, 10-25% squalane, and 100% white tomato extract. White tomatoes are rich in colorless carotenoids, which have excellent antioxidant and whitening properties. They inhibit melanin formation: Colorless carotenoids can inhibit tyrosinase activity, reducing melanin production, and whitening the skin from the root. They also prevent the formation of dark spots: The antioxidant effect can reduce pigmentation caused by ultraviolet rays, preventing and fading dark spots. They promote an even and translucent skin tone: Continuous use can brighten the skin tone and give it a natural glow. This invention primarily relates to the use of white tomato extracts, such as carotenoids, in cosmetics, but does not involve the extraction and application of white tomato peptides.

[0006] Based on the above, the present invention provides a composition containing both leukocyte extract and white tomato polypeptide, which enhances antioxidant and whitening effects through the synergistic effect of the two. This invention aims to propose a new method for applying white tomato polypeptide and leukocyte extract in the field of cosmetics, which is conducive to expanding the application of plant-derived polypeptides. Summary of the Invention

[0007] In view of this, the present invention aims to overcome the shortcomings of the prior art and to provide a composition containing a leukocyte extract and a white tomato extract, and its use in cosmetics. The leukocyte extract is derived from animal umbilical cord blood, thus resolving production and ethical restrictions and enabling scalable production. The leukocyte extract and white tomato polypeptide have good safety and synergistically exhibit significant antioxidant and whitening effects, making them safe for use in the cosmetics field.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present application provides a white tomato polypeptide with antioxidant effect, the amino acid sequence of the polypeptide is YEGDERIREL (SEQ ID NO. 1) and GIEDADPQ (SEQ ID NO. 2).

[0010] Further, the polypeptide is extracted from white tomato (Solanum lycopersicum).

[0011] The present application also provides a preparation method of white blood cell extract, the method comprises the following steps:

[0012] (1) collect 100ml of healthy mammal postpartum umbilical cord blood, inject 10ml of 3.8% w / v sodium citrate anticoagulant, mix evenly, then centrifuge at 3000rpm for 10 minutes, discard the upper plasma after centrifugation, and reserve the lower blood cell layer.

[0013] (2) add physiological saline to the lower blood cell layer at a volume ratio of 1:1, mix evenly, then slowly add to the cell separation liquid, centrifuge at 8000rpm for 15min, after centrifugation, divide into three layers, which are supernatant layer, intermediate white blood cell layer and lower red blood cell layer, and collect the intermediate white blood cell layer.

[0014] (3) centrifuge the collected white blood cell layer at 3000rpm for 10 minutes, discard the supernatant after centrifugation, and the obtained bottom cells are white blood cells.

[0015] (4) inoculate the white blood cells in serum-free medium, add 5% FBS and 300U / ml IL-2, and use the medium to constant volume to about 50ml of culture volume, put it into a 37°, 5% CO2 incubator for culture, according to the cell growth state, supplement appropriate amount of serum-free medium every two days. Culture to 12 days, centrifuge at 8000rpm for 20min to separate and collect all white blood cells.

[0016] (5) add physiological saline to the white blood cells, adjust the concentration of white blood cells in the suspension to 5×10 6 Transfer from room temperature to liquid nitrogen, lyse the white blood cells by repeated freezing and thawing 3 times, centrifuge at 8000rpm for 20min, and collect the supernatant.

[0017] (6) filter the culture solution with 0.22μm sterile filter membrane to remove impurities, the obtained liquid is the supernatant containing white blood cell extract, which is vacuum dried and stored at -80℃ for standby use.

[0018] Further, the animal is selected from sheep, horse, pig, cow, rat, mouse, dog, cat, rabbit, donkey, deer, monkey or human.

[0019] Further, the animal is selected from cow.

[0020] The present application also provides a white tomato polypeptide prepared by the preparation method.

[0021] The present application also provides a white tomato polypeptide preparation method, comprising the following steps:

[0022] (1) Pretreatment

[0023] Fresh white tomatoes are repeatedly washed with flowing clean water to remove impurities such as soil and dust on the surface. After washing, the white tomatoes are placed in a 40-50℃ air drying oven to dry to a moisture content of less than 10%. Then, the dried white tomatoes are crushed using a crusher, and sieved through a 20-40 mesh screen to obtain white tomato powder.

[0024] (2) Homogenization

[0025] According to the solid-liquid ratio 1:1.5 (M / V), deionized water at room temperature is added to the white tomato powder, and soaked for 90 min to make the white tomato powder fully absorb water and swell. Then, according to the mass ratio 1.5%, pectinase is added, and the pH value is adjusted to 4.5 with dilute hydrochloric acid or sodium hydroxide solution. The mixture is placed in a constant temperature water bath at 50℃ for 4h hydrolysis. After hydrolysis is completed, the temperature is increased to 90℃, and the enzyme activity is inactivated for 15 min. The solution is poured into a homogenizer while hot, and homogenized at a pressure of 30 MPa for 2 min to obtain a homogenate.

[0026] (3) Enzymatic hydrolysis

[0027] The pH value of the homogenate is adjusted to 8.0 using an acid-base regulator, and 25000 U / g of papain and 50000 U / g of trypsin are added. The reaction system is placed on a magnetic stirrer at 50℃, and stirred at a speed of 200 r / min for 2h. After the enzymatic hydrolysis reaction is completed, the temperature is increased to 90℃, and the enzyme activity is inactivated for 15 min to obtain an enzymatic hydrolysate.

[0028] (4) Purification I

[0029] The enzymatic hydrolysate is transferred to a centrifuge and centrifuged at a speed of 10000 rpm for 10 min, and the supernatant is collected. The supernatant is separated by ultrafiltration using a membrane filtration system with a molecular weight cutoff of 1 kDa, and the enzymatic hydrolysate is separated by molecular weight to obtain a membrane-separated polypeptide sample solution. The sample solution is then separated by an SP-650M ion exchange column, and then separated and purified by a dextran gel.

[0030] (5) Purification II

[0031] The sample solution is further separated by reverse phase high performance liquid chromatography, the components are collected, dialyzed and desalted, and then lyophilized for use. A high-purity white tomato polypeptide is obtained.

[0032] (6) Polypeptide identification

[0033] The mass spectrometer is used for alternately performing primary mass spectrometry and secondary mass spectrometry on the white tomato polypeptide, and through software analysis, it is found through analysis that the polypeptides with the highest abundance are YEGDERIREL (SEQ ID NO. 1) and GIEDADPQ (SEQ ID NO. 2).

[0034] Further, the chromatographic conditions of the reverse-phase high-performance liquid chromatography are as follows: a C18 chromatographic column (4.6 mm*250 mm, 5 mu m), a mobile phase A of 0.05% trifluoroacetic acid water (V / V), a mobile phase B of acetonitrile, and gradient elution conditions of 0-8 min, 3% B, 8-15 min, 3%-12% B; 15-25 min, 12% B-18% B; 25-30 min, 18% B-30% B; 30-45 min, 30% B-35% B; a flow rate of 1.0 mL / min, and a detection wavelength of 215 nm. The components are collected, dialyzed to remove salt, and freeze-dried for standby, so as to obtain high-purity white tomato polypeptides.

[0035] Further, the polypeptides with the amino acid sequences of SEQ ID NO. 1-2 are entrusted to Shanghai Sangon for synthesis and standby.

[0036] The application also provides a composition comprising the white tomato polypeptide and the white blood cell extract.

[0037] Further, the composition further comprises a cosmetically acceptable adjuvant or carrier.

[0038] Still further, the adjuvant or carrier is selected from at least one of an emulsifier, a co-emulsifier, a skin conditioning agent, a whitening agent, a coloring agent, a humectant, a solubilizer, a surfactant, a preservative, a fragrance, an emollient, an anti-acne agent, a film-forming agent, a thickening agent, a pH regulator, a buffer, a stabilizer, or an ultraviolet absorber.

[0039] The application also provides a cosmetic comprising the composition.

[0040] Further, the cosmetic can further comprise caffeine, and the content refers to the provisions in the Catalogue of Cosmetics Raw Materials (2021 Edition).

[0041] The application also provides the use of the composition in the preparation of a cosmetic with antioxidant and whitening effects.

[0042] Further, the cosmetic includes but is not limited to facial cleanser, skin toner, jelly, emulsion, cream, eye cream, facial mask, essence, aerosol or spray.

[0043] Compared with the prior art, the application has the following beneficial effects:

[0044] The white blood cell extract and the white tomato polypeptide described in SEQ ID NO. 1-2 are good in cell safety, and have certain antioxidant and tyrosinase inhibiting effects. In addition, according to the experimental results of the present application, the white blood cell extract has a significant synergistic effect on the antioxidant and whitening effects of the white tomato polypeptide, and the use after mixing can significantly promote the effects of the two, and can be used for preparing cosmetics with special effects, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0046] Figure 1 Cell toxicity evaluation effect of the white blood cell extract and the white tomato polypeptide of the present application;

[0047] Figure 2 Antioxidant effect of the white blood cell extract and the white tomato polypeptide of the present application;

[0048] Figure 3 Antioxidant effect of the white blood cell extract and the white tomato polypeptide of the present application;

[0049] Figure 4 Tyrosinase inhibiting effect of the white blood cell extract and the white tomato polypeptide of the present application. DETAILED DESCRIPTION

[0050] The following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0051] Example 1

[0052] The present embodiment provides an extraction method of white blood cell extract, comprising the following steps:

[0053] (1) Collect 100ml of healthy postpartum bovine umbilical cord blood, inject 10ml of 3.8% w / v sodium citrate anticoagulant, mix and centrifuge at 3000rpm for 10 minutes, discard the upper plasma after centrifugation, and reserve the lower blood cell layer.

[0054] (2) The lower blood cell layer is mixed with physiological saline at a volume ratio of 1:1, and then slowly added to the cell separation solution. After centrifugation at 8000 rpm for 15 min, the centrifugation is stopped, and the mixture is divided into three layers, i.e., the supernatant layer, the middle white blood cell layer, and the lower red blood cell layer. The middle white blood cell layer is collected.

[0055] (3) The collected white blood cell layer is centrifuged at 3000 rpm for 10 min. After centrifugation, the supernatant is discarded, and the obtained bottom cells are white blood cells.

[0056] (4) The white blood cells are inoculated in a serum-free culture medium, and 5% FBS and 300 U / ml IL-2 are added. The culture medium is adjusted to a culture volume of about 50 ml, and then placed in a 37°, 5% CO2 incubator for culture. According to the growth state of the cells, an appropriate amount of serum-free culture medium is supplemented every two days. After 12 days of culture, the white blood cells are collected by centrifugation at 8000 rpm for 20 min.

[0057] (5) Physiological saline is added to the white blood cells to adjust the concentration of the white blood cells in the suspension to 5×10 6 The white blood cells are transferred from room temperature to liquid nitrogen, and then lysed by repeated freezing and thawing for 3 times. The white blood cells are collected by centrifugation at 8000 rpm for 20 min.

[0058] (6) The culture solution is filtered using a 0.22 μm sterile filter membrane to remove impurities. The obtained liquid is the supernatant containing the white blood cell extract, which is vacuum dried and stored at -80° C for standby use.

[0059] Example 2

[0060] The present embodiment provides a method for extracting white tomato polypeptides, which comprises the following steps:

[0061] (1) Pretreatment

[0062] Fresh white tomatoes are repeatedly washed with flowing clean water to remove impurities such as soil and dust on the surface. After washing, the white tomatoes are placed in a 40° C air-drying oven for drying until the water content is less than 10%. Then, the dried white tomatoes are crushed using a crusher, and passed through a 20-40 mesh screen to obtain white tomato powder.

[0063] (2) Homogenization

[0064] According to the ratio of 1:1.5 (M / V) of the liquid to the white tomato powder, deionized water at room temperature was added, and the white tomato powder was soaked for 90 min to make the white tomato powder fully absorb water and swell. Then, 1.5% of pectinase was added according to the mass ratio, and the pH value was adjusted to 4.5 with dilute hydrochloric acid or sodium hydroxide solution. The mixture was placed in a constant temperature water bath at 50°C for hydrolysis for 4 h. After the hydrolysis was completed, the temperature was increased to 90°C, and the enzyme activity was inactivated for 15 min. The solution was poured into a homogenizer while hot, and homogenized at a pressure of 30 MPa for 2 min to obtain a homogenate.

[0065] (3) Enzymolysis

[0066] The pH value of the homogenate was adjusted to 8.0 using an acid-base regulator, 25,000 U / g of papain and 50,000 U / g of trypsin were added, and the reaction system was placed on a magnetic stirrer at 50°C and stirred at a speed of 200 r / min for 2 h. After the enzyme reaction was completed, the temperature was increased to 90°C, and the enzyme activity was inactivated for 15 min to obtain an enzyme hydrolysate.

[0067] (4) Purification I

[0068] The enzyme hydrolysate was transferred to a centrifuge and centrifuged at a speed of 10,000 rpm for 10 min, and the supernatant was collected. The molecular weight of the enzyme hydrolysate was separated by ultrafiltration separation using a membrane filtration system with a molecular weight cutoff of 1 kDa, and the membrane separation polypeptide sample solution was obtained. The sample solution was then separated by an SP-650M ion exchange column and further purified by a dextran gel.

[0069] (5) Purification II

[0070] Further separation was performed by reverse phase high performance liquid chromatography. The chromatographic conditions were as follows: a C18 chromatographic column (4.6 mm x 250 mm, 5 μm), mobile phase A was 0.05% trifluoroacetic acid water (V / V), mobile phase B was acetonitrile, and the gradient elution conditions were as follows: 0-8 min, 3% B; 8-15 min, 3%-12% B; 15-25 min, 12%-18% B; 25-30 min, 18%-30% B; 30-45 min, 30%-35% B; flow rate was 1.0 mL / min, and detection wavelength was 215 nm. The components were collected, dialyzed, desalted, lyophilized, and stored for use to obtain high-purity white tomato polypeptides.

[0071] (6) Polypeptide identification

[0072] Mass spectrometry was used to alternately perform primary mass spectrometry and secondary mass spectrometry on the white tomato polypeptides. Through software analysis, it was found that the most abundant polypeptides were YEGDERIREL (SEQ ID NO. 1) and GIEDADPQ (SEQ ID NO. 2).

[0073] The white tomato polypeptide with the amino acid sequence SEQ ID NO. 1-2 was commissioned to Shanghai Bioengineering for synthesis and then used.

[0074] Example 3 Cytotoxicity Evaluation

[0075] Test samples: the leukocyte extract obtained in Example 1, and the white tomato polypeptides of SEQ ID NO. 1 and SEQ ID NO. 2 obtained in Example 2. The three test samples were prepared into solutions with a concentration of 0.5 mM.

[0076] Experimental groups: Experimental group 1: 0.5 mM leukocyte extract; Experimental group 2: 0.5 mM polypeptide of SEQ ID NO.1; Experimental group 3: 0.5 mM polypeptide of SEQ ID NO.2; Experimental group 4: equal amounts of 0.5 mM leukocyte extract and 0.5 mM polypeptide of SEQ ID NO.1; Experimental group 5: equal amounts of 0.5 mM leukocyte extract and 0.5 mM polypeptide of SEQ ID NO.2.

[0077] HeLa cells grown to 80% of the bottom area of ​​the culture flask were taken, digested with 0.25% trypsin, and made into a cell density of 1×10 5 / mL of cell suspension. Take 90μL of cell suspension and inoculate it into a 96-well culture plate and place it in an incubator at 37°C and 5% CO2 saturated humidity. After 24 hours of cell culture, aspirate the complete culture medium. Add 10μL of the solution of the above experimental group to each experimental group (each experimental group has 3 replicates), the control group is cells cultured in DMEM medium, and the blank group is DMEM medium without cells, and continue to culture in an incubator at 37°C and 5% CO2 saturated humidity for 24 hours. Add 10μL of CCK-8 reagent to each group, incubate in the cell culture incubator for 2 hours, and use an enzyme-linked immunosorbent assay to measure the absorbance value (OD value) of each well at a wavelength of 450nm. Calculate the cell survival rate according to the following formula based on the mean absorbance of each group:

[0078]

[0079] The experimental results are as follows Figure 1 As shown, the cell viability of each experimental group was above 100%, indicating that the leukocyte extract and white tomato polypeptide prepared by the present invention had no cytotoxicity and good safety.

[0080] Example 4 Antioxidant Activity Determination

[0081] The test products and experimental groups were the same as those in Example 3; 0.5 mM vitamin C was used as a positive control.

[0082] (1) Determination of DPPH free radical scavenging ability

[0083] DPPH ethanol solution with a concentration of 1 x 10 -5 mol / L was prepared and stored in the dark. 2 mL of 0.1 mM DPPH anhydrous ethanol solution was added to a clean test tube containing 2 mL of each experimental group solution, and mixed well. After being placed at room temperature for 30 min, the absorbance was measured at 517 nm. The smaller the absorbance value, the stronger the free radical scavenging ability.

[0084] DPPH free radical scavenging rate (%) = [1-(A i -A j ) / A0] x 100%, wherein A0 is 2 mL of 0.1 mM DPPH anhydrous ethanol solution + 2 mL of pure water, blank control; A i is 2 mL of 0.1 mM DPPH anhydrous ethanol solution + 2 mL of each experimental group solution; A j is 2 mL of anhydrous ethanol + 2 mL of each experimental group solution.

[0085] (2) Determination of ABTS free radical scavenging activity

[0086] ABTS was dissolved in deionized water to a concentration of 7 mmol / L, and potassium persulfate was added to a concentration of 2.45 mmol / L. The solution was then placed in the dark at room temperature overnight for 12-16 h. The generated ABTS free radical solution was diluted with phosphate buffer (PBS, 0.2 mol / L, pH 7.4) to an absorbance value of 0.70 at 734 nm. 0.1 mL of each experimental group solution was taken and mixed with 2.9 mL of ABTS free radical solution, shaken for 30 s, and reacted in the dark for 10 min, then the absorbance value of the reaction solution was measured at 734 nm. Distilled water was used instead of hydrolyzate as a blank.

[0087] ABTS free radical scavenging rate (%) = (A0-A j ) / A0 x 100%, wherein A0 is the absorbance value of 2.9 mL of ABTS reagent mixed with 0.1 mL of distilled water; A j is the absorbance value of 2.9 mL of ABTS reagent mixed with 0.1 mL of hydrolyzate.

[0088] The results of DPPH free radical scavenging ability and ABTS free radical scavenging activity are shown in Tables 1 and 2, respectively. Figures 2-3As shown in Figure 2, the leukocyte extracts and tomato polypeptides of SEQ ID NOs. 1-2 obtained in the present invention both exhibited significant scavenging abilities for DPPH and ABTS free radicals, with the polypeptides exhibiting the best antioxidant activity, reaching a scavenging rate comparable to that of vitamin C. Furthermore, experimental groups 4 and 5 demonstrate that the combined leukocyte extract and white tomato polypeptide exhibited a superior antioxidant effect compared to either the single leukocyte extract or polypeptide alone, demonstrating a significant synergistic effect between the leukocyte extracts and white tomato polypeptides prepared in the present invention.

[0089] Example 5 Tyrosinase Inhibitory Effect

[0090] The test samples and experimental groups were the same as those in Example 3.

[0091] L-tyrosine was saturated and dissolved in PBS buffer solution with an L-tyrosine content of about 0.4 mg / ml. The solution of each experimental group was added and incubated at 37°C for ten minutes. Then, 2 KU units of tyrosinase preheated at 37°C were added. After 24 hours of reaction, the light absorption value of each experimental group at 475 nm was measured. The OD 475nm Represents the content of melanin. Among them, an equal amount of purified water was added as the control group, and the OD of the control group was measured. 475nm The tyrosinase inhibition rate was calculated based on the mean absorbance of each group according to the following formula:

[0092]

[0093] Tyrosinase inhibition effects such as Figure 4 As shown by Figure 4 As can be seen, the leukocyte extract had the lowest tyrosinase inhibition effect, at only 12.4±2.4%, while the white tomato peptide had a superior inhibitory effect. Experimental groups 2-3 and 4-5 show that the leukocyte extract enhanced the enzyme inhibition effect of the white tomato peptide, indicating a synergistic whitening effect between the two.

[0094] In summary of the above embodiments, the leukocyte extract and white tomato polypeptide of the present invention have antioxidant and whitening effects, and are very safe, and can be widely used in the field of cosmetics with antioxidant or whitening effects.

[0095] Based on the above description, it will be understood by those skilled in the art that the present disclosure may be implemented in different specific forms without changing its technical spirit and essential features. Therefore, it should be understood that the above embodiments are not restrictive in all aspects, but illustrative. The scope of the present disclosure is limited by the appended claims, rather than by the preceding description thereof, and therefore all changes and modifications fall within the boundaries and scope of the claims.

Claims

1. A white tomato polypeptide with antioxidant efficacy, characterized in that: The amino acid sequence of the polypeptide is selected from SEQ ID NO. 1-2.

2. A composition, characterized in that The invention comprises the white tomato polypeptide according to any one of claims 1 and a leukocyte extract; the preparation method of the leukocyte extract comprises the following steps: (1) Collect 100 ml of umbilical cord blood from a healthy lactating cow, inject 10 ml of 3.8% w / v sodium citrate for anticoagulation, mix well, and centrifuge at 3000 rpm for 10 minutes. After centrifugation, discard the upper plasma layer and retain the lower blood cell layer. (2) Add physiological saline to the lower blood cell layer at a volume ratio of 1:1, mix well and slowly add it to the cell separation solution, centrifuge at 8000rpm for 15min, and after the centrifugation is completed, it is divided into three layers: the supernatant layer, the middle white blood cell layer and the lower red blood cell layer. Collect the middle white blood cell layer. (3) The collected white blood cell layer was centrifuged at 3000 rpm for 10 minutes. After the centrifugation, the supernatant was discarded and the cells at the bottom were the white blood cells. (4) The leukocytes were inoculated into serum-free medium, and 5% FBS and 300 U / ml IL-2 were added. The volume was adjusted to about 50 ml with the medium and cultured in a 37°C, 5% CO2 incubator. Depending on the cell growth status, an appropriate amount of serum-free medium was added every two days. The culture was terminated after 12 days. All leukocytes were separated and collected by centrifugation at 8000 rpm for 20 minutes. (5) Add physiological saline to the white blood cells to adjust the concentration of white blood cells in the suspension to 5×10 6 The cells were transferred from room temperature to liquid nitrogen, and the leukocytes were lysed by repeated freezing and thawing three times. The cells were centrifuged at 8000 rpm for 20 min, and the supernatant was collected. (6) Filter the culture medium using a 0.22 μm sterile filter membrane to remove impurities. The resulting liquid is the supernatant containing the leukocyte extract. The leukocyte extract is obtained after vacuum drying and stored at -80°C for later use.

3. The composition according to claim 2, characterized in that The composition further comprises auxiliary materials or carriers acceptable in the cosmetic field.

4. The composition according to claim 3, characterized in that The auxiliary material or carrier is selected from at least one of an emulsifier, a co-emulsifier, a skin conditioner, a whitening agent, a colorant, a moisturizer, a solubilizer, a surfactant, a preservative, a fragrance, a moisturizer, an anti-acne agent, a film former, a thickener, a pH adjuster, a buffer, a stabilizer or an ultraviolet absorber.

5. A cosmetic comprising the composition according to any one of claims 2 to 4.

6. Use of the composition according to any one of claims 2 to 4 in the preparation of cosmetics having antioxidant and whitening effects.

7. The use according to claim 6, characterized in that The cosmetics include facial cleansers, toners, gels, lotions, creams, facial masks, essences, and aerosols.

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