Composition containing leukocyte extract and white tomato extract and application of composition in cosmetics

Abstract

The invention relates to a composition containing a leukocyte extract and a white tomato extract and application of the composition in cosmetics, and belongs to the technical field of cosmetics and biology. Specifically, animal umbilical cord blood is selected as a raw material, and the leukocyte extract is obtained through the steps of extraction and purification; meanwhile, white tomato polypeptide is extracted, and further separation is performed to obtain the polypeptide as shown in SEQ ID NO.1-2; the leukocyte extract and the white tomato polypeptide have good safety, have remarkable antioxidant and whitening effects through synergism, can be safely used in the field of cosmetics, and expand the application prospects of the leukocyte extract and the white tomato polypeptide.

Description

Technical Field

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

[0002] Leukocytes are divided into neutrophils, eosinophils, basophils, monocytes, and lymphocytes. They are a very important type of cell in the blood and have the ability to phagocytize foreign substances and produce antibodies, cure body injuries, and resist the invasion of pathogens. Leukocyte extract contains a large amount of cell-specific proteins, lipids, nucleic acids, etc., which can directly act on the mucosal layer, have a high transdermal absorption rate, and have effects such as promoting proliferation and division of fibroblasts, epithelial cells, endothelial cells, promoting angiogenesis, mucosal repair, and cell regeneration. These components can effectively activate aging cells, promote cell renewal, repair inflammatory damage, improve skin immunity, reduce pigmentation, etc., and help the cells in the body restore normal functions and vitality. Therefore, leukocyte extract is often used as an antioxidant drug or added to skin care products. Although leukocyte extract has good biological activity, it cannot play a good role in promoting skin cell proliferation and metabolism. That is to say, directly using leukocyte extract mainly composed of nucleotides in beauty skin care products has unsatisfactory effects. There is a small amount of growth factor in the blood. After extracting leukocytes by traditional extraction methods, the growth factor present in the extract is negligible.

[0003] White tomato (Solanum lycopersicum) is an annual or perennial herbaceous plant of the Solanaceae family and the genus Solanum. Its plant height is 2.5 - 4 meters or has indeterminate growth. The branches have sticky glandular hairs and a strong smell. The stem is prone to lodging. The leaves are pinnate compound leaves or pinnately lobed. The total length of the inflorescence peduncle is 10 - 15 cm, and there are often 6 - 12 flowers. It is an indeterminate growth type tomato, with a single fruit weight of 150g - 200g. After maturity, it becomes golden yellow, has a beautiful appearance, good quality, high fruit hardness, and is resistant to storage. White tomato not only contains nutritional components such as carotene, vitamin C, and B vitamins contained in conventional tomatoes, but also is rich in components such as colorless carotene, phytofluene, phytoene, and lycopene, which have a unique effect on whitening, and the above components are not present or have low content in other color varieties of tomatoes. In addition, the extraction and application of white tomato polypeptide are rarely disclosed in the prior art. It can supplement the collagen required by the human body, and collagen is an important component of cartilage and connective tissue, also helps to maintain skin elasticity and water balance, and at the same time helps to improve the body's resistance and play a role in enhancing immunity.

[0004] CN114191373A discloses a method for quickly and simply extracting leukocyte extract from umbilical cord blood, which comprises the following steps: S1, collection of human umbilical cord blood; S2, centrifuging the umbilical cord blood obtained in step S1 to obtain the supernatant of the buffy coat layer, i.e., umbilical cord blood plasma containing the leukocyte layer, and cryopreserving the plasma; S3, thawing the plasma frozen in step S2, and performing infectious disease antibody detection, viral RT-PCR fluorescence or PCR fluorescence quantitative detection; S4, detecting endotoxin in the plasma; S5, centrifuging the plasma that has passed the detections in steps S3 and S4, performing suction filtration after centrifugation, and finally cryopreserving. This method is for preparing human leukocyte extract from human blood collection, and the yield is small.

[0005] CN119606831A discloses a whitening and repairing composition of white tomatoes, its preparation method and application. By mass percentage, the composition contains: jojoba oil: 68 - 85%, squalane: 10 - 25%, and white tomato extract up to 100%. White tomatoes are rich in colorless carotenoids and have excellent antioxidant and whitening abilities: inhibiting melanin formation: colorless carotenoids can inhibit the activity of tyrosinase and reduce the production of melanin, whitening the skin from the root. Preventing the formation of age spots: the antioxidant effect can reduce the pigment deposition caused by ultraviolet rays, preventing and fading age spots. Uniform and bright skin tone: continuous use can brighten the skin tone and make the skin show a natural luster. This invention mainly relates to the use of white tomato extracts such as carotenoids in cosmetics, but does not involve the extraction and application of white tomato polypeptides.

[0006] Based on the above, the present invention provides a composition containing both leukocyte extract and white tomato polypeptide, and the antioxidant and whitening effects are improved through the synergistic effect of the two, aiming to propose a new method for applying white tomato polypeptide and leukocyte extract to the cosmetic field, which is beneficial to expanding a new direction for the application of plant-derived polypeptides. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to overcome the deficiencies of the prior art. The purpose of the present invention is to provide a composition containing leukocyte extract and white tomato extract and its application in cosmetics. The leukocyte extract is derived from animal umbilical cord blood, so the problems of yield and ethical restrictions can be solved to achieve large-scale production. The leukocyte extract and white tomato polypeptide have good safety, and the two have a significant synergistic antioxidant and whitening effect and can be safely used in the cosmetic field.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] The present invention provides a white tomato polypeptide with antioxidant efficacy, and the amino acid sequences of the polypeptide are 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 invention also provides a method for preparing a white blood cell extract, and the method comprises the following steps:

[0012] (1) Collect 100 ml of healthy mammalian postpartum umbilical cord blood, inject 10 ml of 3.8% w / v sodium citrate for anticoagulation, mix well, centrifuge at 3000 rpm for 10 minutes, discard the upper plasma after centrifugation, and retain the lower blood cell layer.

[0013] (2) Add physiological saline to the lower blood cell layer according to a volume ratio of 1:1, mix well, and slowly add it to the cell separation solution. Centrifuge at 8000 rpm for 15 min. After centrifugation, it is divided into three layers: the upper layer, the middle white blood cell layer, and the lower red blood cell layer. Collect the middle white blood cell layer.

[0014] (3) Centrifuge the collected white blood cell layer at 3000 rpm for 10 minutes. After centrifugation, discard the upper clear liquid, and the resulting bottom cells are white blood cells.

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

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

[0017] (6) Filter the culture solution with a 0.22 μm sterile filter membrane to remove impurities. The resulting liquid is the supernatant containing the white blood cell extract, which is vacuum dried and stored at -80°C for later use.

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

[0019] Still further, the animal is selected from cows.

[0020] The present invention also provides a leukocyte extract obtained by the preparation method.

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

[0022] (1) Pretreatment

[0023] Take fresh white tomatoes, rinse them repeatedly with flowing water to remove impurities such as soil and dust on the surface. After rinsing clean, place the white tomatoes in a blast drying oven at 40 - 50 °C and dry until the moisture content is less than 10%. Subsequently, use a pulverizer to pulverize the dried white tomatoes, and pass them through a 20 - 40 mesh sieve to obtain white tomato powder.

[0024] (2) Homogenization

[0025] Add deionized water at room temperature to the white tomato powder according to the solid - liquid ratio of 1:1.5 (M / V), soak for 90 min to make the white tomato powder fully absorb water and swell. Then, add pectinase according to the mass ratio of 1.5%, adjust the pH value to 4.5 with dilute hydrochloric acid or sodium hydroxide solution, and place the mixture in a constant temperature water bath at 50 °C for hydrolysis for 4 h. After the hydrolysis is completed, raise the temperature to 90 °C and keep it warm for 15 min to inactivate the enzyme. Pour the solution into a homogenizer while it is hot and homogenize it at a pressure of 30 MPa for 2 min to obtain a homogenate.

[0026] (3) Enzymolysis

[0027] Use an acid - base regulator to adjust the pH value of the homogenate to 8.0, add papain at an addition amount of 25000 U / g and trypsin at an addition amount of 50000 U / g, place the reaction system on a magnetic stirrer at 50 °C, and stir and react at a rotation speed of 200 r / min for 2 h. After the enzymolysis reaction is completed, raise the temperature to 90 °C and keep it warm for 15 min to inactivate the enzyme, and obtain an enzymolysis solution.

[0028] (4) Purification I

[0029] Transfer the enzymolysis solution to a centrifuge, centrifuge at a rotation speed of 10000 rpm for 10 min, and collect the supernatant. Use a membrane filtration system with a cut - off molecular weight of 1 kDa for ultrafiltration separation, separate the enzymolysis products according to molecular weight to obtain a membrane - separated polypeptide sample solution, and then separate it through an SP - 650M ion exchange column and further purify it with Sephadex.

[0030] (5) Purification II

[0031] Perform further separation by reverse - phase high - performance liquid chromatography, collect the components, dialyze to remove salt, and freeze - dry for standby to obtain high - purity white tomato polypeptide.

[0032] (6) Polypeptide identification

[0033] The white tomato polypeptides were alternately subjected to first - stage mass spectrometry and second - stage mass spectrometry using a mass spectrometer. With the aid of software analysis, it was found that the polypeptides with the highest abundances were YEGDERIREL (SEQ ID NO.1) and GIEDADPQ (SEQ ID NO.2).

[0034] Furthermore, the chromatographic conditions for reverse - phase high - performance liquid chromatography were as follows: a C18 chromatographic column (4.6 mm×250 mm, 5 μm), mobile phase A was 0.05% trifluoroacetic acid in water (V / V), mobile phase B was acetonitrile, and the gradient elution conditions were: 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; the flow rate was 1.0 mL / min, and the detection wavelength was 215 nm. The collected fractions were dialyzed to remove salts and freeze - dried for later use to obtain high - purity white tomato polypeptides.

[0035] Furthermore, the polypeptides with amino acid sequences SEQ ID NO.1 - 2 were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and then reserved for later use.

[0036] The present invention also provides a composition, which comprises the above - mentioned white tomato polypeptides and the above - mentioned white blood cell extract.

[0037] Furthermore, the composition further comprises excipients or carriers acceptable in the cosmetic field.

[0038] Even further, the excipients or carriers are selected from at least one of emulsifiers, co - emulsifiers, skin conditioners, whitening agents, colorants, moisturizers, solubilizers, surfactants, preservatives, fragrances, emollients, anti - acne agents, film - forming agents, thickeners, pH regulators, buffers, stabilizers or ultraviolet absorbers.

[0039] The present invention also provides a cosmetic comprising the above - mentioned composition.

[0040] Furthermore, the cosmetic may further include caffeine, and the content refers to the provisions of the "List of Cosmetic Raw Materials in Use (2021 Edition)".

[0041] The present invention also provides the application of the above - mentioned composition in the preparation of cosmetics with antioxidant and whitening effects.

[0042] Furthermore, the cosmetics include, but are not limited to, facial cleansers, toners, gels, lotions, creams, eye creams, facial masks, essences, aerosols or sprays.

[0043] The present invention has the following beneficial effects compared with the prior art:

[0044] The present invention prepares and obtains a leukocyte extract and the white tomato polypeptides described in SEQ ID NO.1-2. The leukocyte extract and the white tomato polypeptides have good cell safety, and have certain antioxidant and tyrosinase inhibitory effects. In addition, from the experimental results of the present invention, it can be seen that the leukocyte extract has a significant synergistic effect on the antioxidant and whitening effects of the white tomato polypeptides. Using them in combination can significantly promote the efficacy of both, and they can be used to prepare cosmetics with special effects, and have a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0046] Figure 1 Cytotoxicity evaluation effect of the leukocyte extract and the white tomato polypeptides of the present invention;

[0047] Figure 2 Antioxidant effect of the leukocyte extract and the white tomato polypeptides of the present invention;

[0048] Figure 3 Antioxidant effect of the leukocyte extract and the white tomato polypeptides of the present invention;

[0049] Figure 4 Tyrosinase inhibitory effect of the leukocyte extract and the white tomato polypeptides of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0050] The following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention fall within the scope of protection of the present invention.

[0051] Example 1

[0052] This example provides a method for extracting a leukocyte extract, including the following steps:

[0053] (1) Collect 100 ml of healthy postpartum bovine umbilical cord blood, 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.

[0054] (2) Add physiological saline to the lower layer of blood cells at a volume ratio of 1:1. After mixing evenly, slowly add it to the cell separation solution and centrifuge at 8000 rpm for 15 minutes. After centrifugation, it is divided into three layers: the upper layer, the middle layer of white blood cells, and the lower layer of red blood cells. Collect the middle layer of white blood cells.

[0055] (3) Centrifuge the collected white blood cell layer at 3000 rpm for 10 minutes. After centrifugation, discard the upper clear liquid, and the cells at the bottom are white blood cells.

[0056] (4) Inoculate white blood cells into a serum-free medium, add 5% FBS and 300 U / ml of IL-2, and make up the volume of the medium to about 50 ml with the medium. Place it in an incubator at 37°C and 5% CO2 for culture. According to the cell growth status, supplement an appropriate amount of serum-free medium every two days. After culturing for 12 days, end the culture and centrifuge at 8000 rpm for 20 minutes to separate and collect all white blood cells.

[0057] (5) Add physiological saline to white blood cells to adjust the concentration of white blood cells in the suspension to 5×10 6 cells / mL. Transfer from room temperature to liquid nitrogen and lyse white blood cells by repeated freezing and thawing 3 times. Centrifuge at 8000 rpm for 20 minutes and collect the supernatant.

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

[0059] Example 2

[0060] This example provides a method for extracting white tomato polypeptides, including the following steps:

[0061] (1) Pretreatment

[0062] Take fresh white tomatoes and rinse them repeatedly with flowing water to remove impurities such as soil and dust on the surface. After rinsing clean, place the white tomatoes in a blast drying oven at 40°C and dry them until the moisture content is less than 10%. Subsequently, use a pulverizer to pulverize the dried white tomatoes and pass them through a 20-40 mesh sieve to obtain white tomato powder.

[0063] (2) Homogenization

[0064] Add deionized water at room temperature to white tomato powder according to the solid-liquid ratio of 1:1.5 (M / V), soak for 90 min to allow the white tomato powder to fully absorb water and swell. Then, add pectinase according to the mass ratio of 1.5%, adjust the pH value to 4.5 with dilute hydrochloric acid or sodium hydroxide solution, and hydrolyze the mixture in a constant temperature water bath at 50 °C for 4 h. After hydrolysis, raise the temperature to 90 °C and keep warm for 15 min to inactivate the enzyme. Pour the solution into a homogenizer while it is hot and homogenize it at a pressure of 30 MPa for 2 min to obtain a homogenized liquid.

[0065] (3) Enzymatic hydrolysis

[0066] Adjust the pH value of the homogenized liquid to 8.0 using an acid-base regulator, add papain at an addition amount of 25000 U / g and trypsin at an addition amount of 50000 U / g, place the reaction system on a magnetic stirrer at 50 °C, and stir and react at a rotation speed of 200 r / min for 2 h. After the enzymatic hydrolysis reaction ends, raise the temperature to 90 °C and keep warm for 15 min to inactivate the enzyme to obtain an enzymatically hydrolyzed solution.

[0067] (4) Purification I

[0068] Transfer the enzymatically hydrolyzed solution to a centrifuge, centrifuge at a rotation speed of 10000 rpm for 10 min, and collect the supernatant. Use a membrane filtration system with a cut-off molecular weight of 1 kDa for ultrafiltration separation, separate the enzymatic hydrolysis products using molecular weight, obtain a membrane separation polypeptide sample solution, and then separate it through an SP-650M ion exchange column and further purify it using Sephadex.

[0069] (5) Purification II

[0070] Use reverse-phase high performance liquid chromatography for further separation. Chromatographic conditions: C18 chromatographic column (4.6 mm × 250 mm, 5 μm), mobile phase A is 0.05% trifluoroacetic acid water (V / V), mobile phase B is acetonitrile, and the gradient elution conditions are: 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; the flow rate is 1.0 mL / min, and the detection wavelength is 215 nm. Collect the components, dialyze to remove salts, freeze-dry for standby to obtain high-purity white tomato polypeptide.

[0071] (6) Polypeptide identification

[0072] Use a mass spectrometer to alternately perform primary mass spectrometry and secondary mass spectrometry on white tomato polypeptide, and analyze with the help of software. It is found through analysis that the polypeptides with the highest abundances are YEGDERIREL (SEQ ID NO.1) and GIEDADPQ (SEQ ID NO.2).

[0073] The white tomato polypeptides with amino acid sequences SEQ ID NO.1-2 were commissioned to Shanghai Sangon for synthesis and then reserved for use.

[0074] Example 3 Cytotoxicity Evaluation

[0075] Test articles: The white blood cell 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 above three test articles were respectively prepared into solutions with a concentration of 0.5 mM.

[0076] Experimental grouping: Experimental group 1: 0.5 mM white blood cell 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 white blood cell extract and 0.5 mM polypeptide of SEQ ID NO.1; Experimental group 5: equal amounts of 0.5 mM white blood cell extract and 0.5 mM polypeptide of SEQ ID NO.2.

[0077] Take HeLa cells that have grown to 80% of the bottom area of the culture flask, digest them with 0.25% trypsin, and make a cell suspension with a cell density of 1×10 5 cells / mL with complete culture medium. Take 90 μL of the 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 for culture. After 24 h of cell culture, aspirate the complete culture medium. Add 10 μL of the solutions in the above experimental groupings to each experimental group (each experimental group has 3 replicates), the control group is cells cultured with DMEM medium, and the blank group is DMEM medium without cells. Continue to culture in an incubator at 37 °C and 5% CO2 saturated humidity for 24 h. Add 10 μL of CCK-8 reagent to each group, incubate in the cell culture incubator for 2 h, and use an enzyme-linked immunosorbent detector to measure the absorbance value (OD value) of each well at a wavelength of 450 nm. Calculate the cell survival rate according to the average absorbance of each group according to the following formula:

[0078]

[0079] The experimental results are as Figure 1 shown. The cell viability of each experimental group is above 100%, indicating that both the white blood cell extract and the white tomato polypeptides prepared by the present invention have no cytotoxicity and good safety.

[0080] Example 4 Determination of Antioxidant Activity

[0081] Test articles and experimental grouping are the same as in Example 3; 0.5 mM vitamin C is used as a positive control.

[0082] (1) Determination of DPPH Free Radical Scavenging Ability

[0083] Prepare a 1×10 -5 mol / L DPPH ethanol solution and store it in the dark. Add 2 mL of 0.1 mM DPPH anhydrous ethanol solution to a clean test tube containing 2 mL of each experimental group solution and mix well. After standing at room temperature for 30 min, measure the absorbance 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] × 100%, where A0 is the absorbance of a blank control consisting of 2 mL of 0.1 mM DPPH anhydrous ethanol solution + 2 mL of pure water; A i is the absorbance of 2 mL of 0.1 mM DPPH anhydrous ethanol solution + 2 mL of each experimental group solution; A j is the absorbance of 2 mL of anhydrous ethanol + 2 mL of each experimental group solution.

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

[0086] Dissolve ABTS in deionized water to make the ABTS concentration reach 7 mmol / L, and add potassium persulfate to make the concentration of potassium persulfate 2.45 mmol / L. Then place the solution in the dark at room temperature overnight for 12 - 16 h. Dilute the generated ABTS free radical solution with phosphate buffer (PBS, 0.2 mol / L, pH 7.4) to make its absorbance value at 734 nm be 0.70. Take 0.1 ml of each experimental group solution and mix it with 2.9 ml of ABTS free radical solution, shake well for 30 s, react in the dark for 10 min, and then measure the absorbance value of the reaction solution at 734 nm. Use distilled water instead of the hydrolysis solution as the blank.

[0087] ABTS free radical scavenging rate (%) = (A0 - A j ) / A0 × 100%, where A0 is the absorbance value of the mixture of 2.9 mL ABTS reagent and 0.1 mL distilled water; A j is the absorbance value of the mixture of 2.9 mL ABTS reagent and 0.1 mL hydrolysis solution.

[0088] The results of DPPH free radical scavenging ability and ABTS free radical scavenging activity are respectively as Figures 2-3As shown. The leukocyte extract extracted by the present invention and the tomato polypeptides of SEQ ID NO.1-2 obtained both have significant ability to scavenge DPPH and ABTS free radicals. Among them, the antioxidant effect of the polypeptide is the best, and the scavenging rate is consistent with that of vitamin C. In addition, it can be seen from Experimental Groups 4-5 that the antioxidant effect of the mixture of leukocyte extract and white tomato polypeptide is better than that of the single leukocyte extract or polypeptide, indicating that the leukocyte extract and white tomato polypeptide prepared by the present invention have a significant synergistic effect.

[0089] Example 5 Inhibitory Effect on Tyrosinase

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

[0091] L-tyrosine was saturated and dissolved in PBS buffer with the L-tyrosine content of about 0.4 mg / ml. The solution of each experimental group was added respectively. After incubating at 37 °C for ten minutes, 2 KU units of tyrosinase preheated at 37 °C were added to each. After reacting for 24 hours, the light absorption values of each experimental group at 475 nm were measured respectively. This 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 . According to the average absorbance of each group, the tyrosinase inhibition rate was calculated according to the following formula:

[0092]

[0093] The inhibitory effect on tyrosinase is as Figure 4 shown. It can be seen from Figure 4 that the inhibitory effect of the leukocyte extract on tyrosinase is the lowest, only 12.4 ± 2.4%, while the inhibitory effects of the white tomato polypeptides are better than that of the leukocyte extract. According to Experimental Groups 2-3 and Experimental Groups 4-5, the leukocyte extract can promote the enzyme inhibitory effect of the white tomato polypeptide, indicating that there is a synergistic whitening effect between the two.

[0094] Based on the above examples, the leukocyte extract and white tomato polypeptide of the present invention have antioxidant and whitening effects, and have good safety, and can be widely used in the cosmetic field with antioxidant or whitening effects.

[0095] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing its technical spirit and essential features. Therefore, it should be understood that the above embodiments are illustrative in all aspects rather than restrictive. The scope of the present disclosure is limited by the appended claims rather than by the description preceding them, and thus 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 method for preparing a leukocyte extract, characterized in that, Comprising the following steps: (1) Collect 100 ml of postpartum umbilical cord blood from healthy mammals, inject 10 ml of 3.8% w / v sodium citrate for anticoagulation, mix well, centrifuge at 3000 rpm for 10 minutes, discard the upper plasma after centrifugation, 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 8000 rpm for 15 min. After centrifugation, it is divided into three layers: the upper layer, the middle white blood cell layer, and the lower red blood cell layer. Collect the middle white blood cell layer; (3) Centrifuge the collected white blood cell layer at 3000 rpm for 10 minutes. After centrifugation, discard the upper clear liquid, and the cells at the bottom are white blood cells; (4) Inoculate white blood cells into a serum-free medium, add 5% FBS and 300 U / ml of IL-2, and make up the volume of the medium to about 50 ml with the medium. Place it in a 37° and 5% CO2 incubator for culture. According to the cell growth state, supplement an appropriate amount of serum-free medium every two days. After culturing for 12 days, end the culture, and centrifuge at 8000 rpm for 20 min to separate and collect all white blood cells; (5) Add physiological saline to the white blood cells and adjust the concentration of white blood cells in the suspension to 5×10 6 cells / mL. Transfer from room temperature to liquid nitrogen, lyse the white blood cells by repeated freezing and thawing 3 times, centrifuge at 8000 rpm for 20 min, and collect the supernatant; (6) Filter the culture solution with a 0.22 μm sterile filter membrane to remove impurities. The obtained liquid is the supernatant containing the white blood cell extract. After vacuum drying, the white blood cell extract is obtained and stored at -80 °C for later use.

3. The preparation method according to claim 2, characterized in that, The animal is selected from sheep, horse, pig, cow, rat, mouse, dog, cat, rabbit, donkey, deer, monkey or human; preferably, the animal is selected from cows.

4. The white blood cell extract prepared by the preparation method according to any one of claims 2-3.

5. A composition, characterized in that, Comprising the white tomato polypeptide according to any one of claim 1 and the white blood cell extract according to any one of claims 2-3.

6. The composition according to claim 5, wherein The composition further comprises excipients or carriers acceptable in the cosmetic field.

7. The composition according to claim 6, characterized in that, The excipients or carriers are selected from at least one of emulsifiers, co-emulsifiers, skin conditioners, whitening agents, colorants, moisturizers, solubilizers, surfactants, preservatives, fragrances, emollients, anti-acne agents, film formers, thickeners, pH regulators, buffers, stabilizers or ultraviolet absorbers.

8. A cosmetic comprising the composition according to any one of claims 5-7.

9. Use of the composition according to any one of claims 5-7 in the preparation of a cosmetic having antioxidant and whitening effects.

10. The application according to claim 9, wherein The cosmetics include facial cleanser, toner, gel, lotion, cream, eye cream, mask, essence, aerosol or spray.

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