Application of recombinant human interleukin 2 (I) in the preparation of anti-wrinkle and firming products

Abstract

The invention belongs to the field of biomedicine technology, and specifically relates to an application of recombinant human interleukin 2 (I) in anti-wrinkle and firming products. The anti-wrinkle and firming products include cosmetics. The recombinant human interleukin 2 (I) is added to a common carrier of cosmetics to produce anti-wrinkle and firming products. The recombinant human interleukin 2 (I) can significantly improve the elastin content of fibroblasts induced by long-wave dark spot effect ultraviolet radiation within a concentration range of 1.25%-5% (v / v), has anti-wrinkle and firming effects, and does not show fibroblast toxicity in an in vitro cytotoxicity test, and is a safe and effective new anti-wrinkle and firming ingredient. The production of the recombinant human interleukin 2 (I) specifically includes seed culture and fermentation, bacterial cell collection, inclusion body preparation and dissolution, crude extraction, desalting and renaturation, reverse high-pressure liquid phase refining, removal of mobile phase and stock solution filtration and sterilization, semi-finished product preparation and finished product packaging.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of recombinant human interleukin 2 (I) in the preparation of anti-wrinkle and firming products. Background Art

[0002] Recombinant human interleukin 2 (I) is a cytokine with immunomodulatory function, which can promote the proliferation and differentiation of B cells, promote the rapid proliferation of T cells, and induce the differentiation and proliferation of NK cells, thereby improving the body's immunity. It can be used for tumor immunotherapy as an auxiliary means of tumor treatment. On the one hand, it adjusts and enhances the body's immune function, and on the other hand, it enhances the ability of various killer cells in the body to destroy tumor cells. The recombinant human interleukin 2 (I) currently on the market is mainly recombinant human interleukin 2 (I) expressed by genetically engineered bacteria. Through genetically engineered bacterial fermentation and appropriate separation and purification technology, a high-purity stock solution is obtained, and then made into a finished product.

[0003] As the largest and most important organ of the human body, the skin is in direct contact with the external environment and has the functions of protection, excretion, regulating body temperature and sensing external stimuli. Within 24 hours a day, the skin state will show obvious circadian rhythms as time goes by and day and night pass. Factors such as climate change, air pollution, work and life pressure, irregular diet, endocrine disorders and improper care can change the biological rhythm of the skin, resulting in the deterioration of the skin's daytime barrier function and nighttime repair function, resulting in dryness, sagging and aging. Aging is a natural phenomenon that humans must experience in their lives. Skin aging is mainly manifested in wrinkle formation, wrinkles, sagging and roughness. The causes of skin aging are very complex and can be divided into exogenous aging and endogenous aging. Exogenous aging mainly refers to environmental factors such as ultraviolet radiation, smoking, wind, sun exposure and exposure to harmful chemicals. Among them, since ultraviolet radiation in sunlight is the main factor causing skin aging among environmental factors, exogenous aging is also called photoaging. Natural aging is caused by internal factors, including: aging, physiological aging, decreased collagen and elastin synthesis in the skin, decreased cytoplasmic and intercellular fluidity. Specific manifestations: dry skin, skin atrophy, elastic tissue degeneration, thinning skin and decreased sebaceous gland secretion.

[0004] In order to improve skin wrinkles, people try to delay aging by increasing the collagen content in the skin and reducing free radicals in the body. In addition to traditional anti-aging ingredients (peptides, vitamin C, hyaluronic acid and regenerative cytokines), more and more plant extracts are used for anti-aging. However, since most plant extracts are mixed ingredients, their efficacy and whether there is a synergistic effect between multiple ingredients are still unknown, which restricts their application. According to the test of a third-party testing agency, using fibroblasts as the test system, the recombinant human interleukin-2 (I) stock solution showed a significant increase in the elastin content of fibroblasts induced by long-wave dark spot effect ultraviolet radiation, with anti-wrinkle and firming effects, and no fibroblast toxicity was shown in in vitro tests. It is a safe and effective new anti-wrinkle and firming ingredient, which may have broad application prospects in the fields of cosmetics, medical beauty, etc. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention develops a recombinant human interleukin 2 (I) for use in the preparation of anti-wrinkle and firming products, which can significantly increase the elastin content of fibroblasts induced by long-wave dark spot effect ultraviolet radiation, has anti-wrinkle and firming effects, and does not show toxicity to human fibroblasts in in vitro tests. It is a safe and effective new anti-wrinkle and firming ingredient.

[0006] The technical solution of the present invention to solve the technical problem is: an application of recombinant human interleukin 2 (I) in the preparation of anti-wrinkle and firming products, wherein the recombinant human interleukin 2 (I) is used as an active component of the anti-wrinkle and firming products, and the volume concentration of the recombinant human interleukin 2 (I) is 1.25%-5%;

[0007] The production method of recombinant human interleukin-2 (I) comprises the following steps:

[0008] (1) Seed culture and fermentation: Take the recombinant human interleukin-2 (I) working seeds and inoculate them into the culture medium for culture. After the culture is completed, inoculate them into the fermentation tank for fermentation;

[0009] (2) Collecting bacteria: remove the supernatant by centrifugation and collect the bacteria;

[0010] (3) Preparation and dissolution of inclusion bodies: The bacterial suspension is broken with a high-pressure homogenizer, washed to remove some bacterial fragments and cell impurities, centrifuged to obtain the precipitate, and dissolved to obtain the inclusion body solution;

[0011] (4) Crude extraction: The inclusion body solution is loaded and eluted to remove large molecular proteins and collect the target protein;

[0012] (5) Desalting and renaturation: Collect the desalted protein solution, dilute it, add a catalyst, and oxidize and renature it to restore the protein's higher-order structure to obtain a renaturation solution;

[0013] (6) Reverse high-performance liquid phase purification: The refolded liquid is further purified by a reversed-phase high-performance liquid phase system to remove impurities and obtain a high-purity sample solution;

[0014] (7) Removing the mobile phase and filtering and sterilizing the stock solution: The sample solution collected by the HPLC is placed in a freeze dryer, and the organic solvent is removed by freeze drying. The sample is white and loose. The white and loose sample is dissolved with a buffer solution. After the solution is dissolved, it is the recombinant human interleukin-2 (I) stock solution;

[0015] (8) Preparation of semi-finished products: Clean the liquid preparation and filtration system and two-stage filter cartridges, sterilize online with high-pressure steam, collect the excipients according to the amount indicated in the instruction sheet, dissolve them with injection water to make up the volume, slowly pour the recombinant human interleukin-2 (I) stock solution into the liquid preparation tank, stir and mix with the excipient solution, and sterilize and filter through a two-stage filter;

[0016] (9) Packaging of finished products.

[0017] Preferably, the centrifugation operation in step (2) adopts a continuous flow centrifuge.

[0018] Preferably, the elution operation in step (4) adopts an propylene glycol S-200 chromatography system.

[0019] Preferably, in the step (5), the desalted protein solution is collected by loading the solution onto a Sephadex G25 chromatography system; and the catalyst is cupric chloride.

[0020] Preferably, the buffer solution in step (7) is sterilized and filtered using a filter membrane under a clean bench.

[0021] Preferably, the auxiliary materials in step (8) include phosphate, sodium dodecyl sulfate, human albumin and mannitol.

[0022] Preferably, the finished product packaging in step (9) includes: cleaning and sterilizing the rubber stopper, cleaning and sterilizing the vial, sterilizing the pot cover, taking out the sterilized canned parts from the sterilizer under the protection of Class A laminar flow, aseptically assembling them in sequence, starting normal canning after the filling volume is debugged and qualified, semi-stoppering, and transferring the canned product to the freeze dryer through the transfer system for freeze drying. After the freeze drying is completed, the product is stoppered and then capped.

[0023] Preferably, the anti-wrinkle and firming product comprises cosmetics, and the recombinant human interleukin-2 (I) is added to a common cosmetic carrier to prepare the anti-wrinkle and firming product, and the common carrier is a combination of one or more of glycerol, vegetable oil, mineral oil, liposome, silicone oil and water.

[0024] The effects provided in the content of the invention are only the effects of the embodiments, rather than all the effects of the invention. The above technical solution has the following advantages or beneficial effects:

[0025] The invention has anti-wrinkle and firming effects within the range of 1.25%-5% (v / v) recombinant human interleukin 2 (I) concentration, and does not show human fibroblast toxicity in in vitro tests. It is a safe and effective new anti-wrinkle and firming ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the cell viability graph;

[0027] Figure 2 This is a trend chart of elastin content changes. DETAILED DESCRIPTION

[0028] In order to clearly illustrate the technical features of the present invention, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings.

[0029] Example 1

[0030] A use of recombinant human interleukin 2 (I) in the preparation of an anti-wrinkle and firming product, wherein the recombinant human interleukin 2 (I) is used as an active component of the anti-wrinkle and firming product, wherein the volume concentration of the recombinant human interleukin 2 (I) is 1.25%-5%. Within the volume concentration range of 1.25%-5%, the recombinant human interleukin 2 (I) significantly increases the elastin content of fibroblasts induced by long-wave dark spot effect ultraviolet radiation and does not show fibroblast toxicity.

[0031] The production method of recombinant human interleukin-2 (I) comprises the following steps:

[0032] (1) Seed culture and fermentation: Take the recombinant human interleukin-2 (I) working seeds and inoculate them into the culture medium for culture. After the culture is completed, inoculate them into the fermentation tank for fermentation;

[0033] (2) Collecting bacteria: remove the supernatant by centrifugation and collect the bacteria;

[0034] (3) Preparation and dissolution of inclusion bodies: The bacterial suspension is broken with a high-pressure homogenizer, washed to remove some bacterial fragments and cell impurities, centrifuged to obtain the precipitate, and dissolved to obtain the inclusion body solution;

[0035] (4) Crude extraction: The inclusion body solution is loaded and eluted to remove large molecular proteins and collect the target protein;

[0036] (5) Desalting and renaturation: Collect the desalted protein solution, dilute it, add the catalyst, and oxidize and renature it for 3 hours to restore the protein's higher-order structure to obtain the renaturation solution;

[0037] (6) Reverse high-performance liquid phase purification: The refolded liquid is further purified by a C18 reverse-phase high-performance liquid phase system to remove impurities and obtain a high-purity sample solution;

[0038] (7) Removing the mobile phase and filtering and sterilizing the stock solution: Put the sample solution collected by the HPLC into a freeze dryer and remove the organic solvent by freeze drying. The sample is white and loose. Dissolve the white and loose sample with buffer. The dissolved sample is the recombinant human interleukin-2 (I) stock solution and is stored at 2-8°C.

[0039] (8) Preparation of semi-finished products: Clean the liquid preparation and filtration system and two-stage filter cartridges, sterilize online with high-pressure steam, collect the excipients according to the amount indicated in the instruction sheet, dissolve them with injection water to make up the volume, slowly pour the recombinant human interleukin-2 (I) stock solution into the liquid preparation tank, stir and mix with the excipient solution, and sterilize and filter through a two-stage filter;

[0040] (9) Packaging of finished products.

[0041] The centrifugation operation in step (2) adopts a continuous flow centrifuge.

[0042] The elution operation in step (4) adopts an propylene dextran gel S-200 chromatography system.

[0043] The specific operation of collecting the desalted protein solution in step (5) is to load the solution onto a Sephadex G25 chromatography system for collection; the catalyst is cupric chloride.

[0044] The buffer solution in step (7) is sterilized and filtered using a 0.2 μm filter membrane under a clean bench.

[0045] The auxiliary materials in step (8) include phosphate, sodium lauryl sulfate, human albumin and mannitol.

[0046] The finished product packaging in step (9) includes: cleaning and sterilizing the rubber stopper, cleaning and sterilizing the vial, sterilizing the pot cover, taking out the sterilized canned parts from the sterilizer under the protection of Class A laminar flow, aseptically assembling them in sequence, starting normal canning after the filling volume is debugged and qualified, semi-pressing the stopper, and transferring the canned product to the freeze dryer through the transfer system for freeze drying. After the freeze drying is completed, the product is pressed and capped.

[0047] The anti-wrinkle and firming product comprises cosmetics. The recombinant human interleukin-2 (I) is added to a common carrier of cosmetics to prepare the anti-wrinkle and firming product. The common carrier is a combination of one or more of glycerol, vegetable oil, mineral oil, liposome, silicone oil and water.

[0048] Example 2

[0049] In vitro cytotoxicity assay:

[0050]

[0051] 1. Purpose of testing

[0052] This test uses the MTT method to detect cytotoxicity and evaluates the toxicity of the test sample to fibroblasts by cell survival rate.

[0053] 2. Main Reagents

[0054] High glucose DMEM culture medium, fetal bovine serum, phosphate buffered saline, thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide and trypsin.

[0055] 3. Main equipment

[0056] CO2 incubator, biological safety cabinet, inverted microscope and microplate reader.

[0057] 4. Detection Methods

[0058] Cell seeding: 8×10 3 The cells were inoculated into a 96-well plate at a density of 1000 cells / well and incubated overnight in an incubator (37°C, 5% carbon dioxide).

[0059] Experimental grouping: set up zero adjustment group, control group, positive control group and sample group. In the sample group, 10% concentration sample corresponds to 110μg / mL, then half diluted, 8 concentration gradients are set for the sample, and 3 replicate wells are set for each concentration gradient.

[0060] Solution preparation: Prepare sample working solutions of different concentrations according to the detection concentration setting table.

[0061] Table 1: Detection concentration setting table

[0062]

[0063] Dosing: Dosing was performed when the cell plating rate in the 96-well plate reached 40%-60%. 200 μL of culture medium containing 10% phosphate buffer was added to each well of the control group; 200 μL of culture medium containing 10% dimethyl sulfoxide was added to each well of the positive control group; 200 μL of culture medium containing samples of corresponding concentrations was added to each well of the sample group; no cells were inoculated in the zero adjustment group, and only 200 μL of cell culture medium was added. After the administration was completed, the 96-well plate was placed in an incubator (37°C, 5% carbon dioxide) for incubation.

[0064] Detection: After incubating the cells for 24 hours, discard the supernatant, add thiazolyl blue tetrazolium bromide (MTT) working solution (0.5 mg / mL), and incubate at 37°C in the dark for 2 hours. After the incubation, discard the supernatant, add 100 μL of dimethyl sulfoxide to each well, and read the OD (optical density) value at 490 nm.

[0065] Calculation of relative cell viability: Calculated according to the following formula

[0066]

[0067] 5. Cytotoxicity test results

[0068] The samples were set at 8 dosing concentrations and cytotoxicity tests were performed. The MTT test results are as follows:

[0069] Table 2: MTT assay results

[0070]

[0071] The cell relative viability graph was drawn with the eight selected sample concentrations as the horizontal axis and the cell relative viability values ​​as the vertical axis (see Figure 1 According to the results of MTT test, the samples showed no fibroblast toxicity within the concentration range of 5% (v / v).

[0072] like Figure 1 Shown is a graph of cell viability.

[0073] 6. Conclusion

[0074] Under the conditions of this assay, the sample recombinant human interleukin-2 (I) showed no fibroblast toxicity within the concentration range of 5% (v / v).

[0075] Example 3

[0076] Long-wave ultraviolet light stimulates human fibroblasts

[0077]

[0078] 1. Test Purpose

[0079] This test uses fibroblasts as the test system and evaluates whether the sample to be tested has anti-wrinkle and firming effects based on the content of the main types of elastin.

[0080] 2. Main Reagents

[0081] High glucose DMEM culture medium, fetal bovine serum, phosphate buffered saline, thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide and trypsin.

[0082] 3. Main equipment

[0083] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader and floor-standing half-body UV therapy device.

[0084] 4. Test Methods

[0085] The specific settings of the experimental groups are shown in Table 3.

[0086] Table 3: Experimental design

[0087]

[0088] Cell seeding: according to the appropriate seeding density (8×10 4 Cells were seeded into 24-well plates at 100 cells / well and incubated overnight in an incubator (37°C, 5% carbon dioxide).

[0089] Experimental grouping: The experiment set up a blank control group, a negative control group, a positive control group and a sample group. The sample group was set up with 3 concentration gradients.

[0090] Solution preparation: Prepare working solutions of the test substance of different concentrations according to the test concentration design table.

[0091] Dosing: Dosing was performed when the cell plating rate in the 24-well plate reached 40%-60%. 1 mL of cell culture medium was added to each well of the blank control group and the negative control group; 1 mL of culture medium containing 100 μg / mL vitamin C and 7 μg / mL vitamin E was added to each well of the positive control group; 1 mL of culture medium containing the test substance at the corresponding concentration was added to each well of the sample group.

[0092] Radiation: 24 hours after administration, the total dose received by the negative control group, positive control group and sample group was 9J / cm 2 The blank control group was placed in the same environment (the long-wave dark spot effect ultraviolet radiation dose was 0J / cm 2 ).

[0093] Collecting cell supernatant: After incubation for 24 hours, collect the cell culture supernatant in a centrifuge tube and store it in a -80°C refrigerator.

[0094] Elastin detection: The elastin content was detected and analyzed according to the operating instructions of the enzyme-labeled antibody detection kit.

[0095] 5. Test results

[0096] According to the specific experimental method, the elastin content was tested. The test results are shown in the following table. The change trend is as follows: Figure 2 shown.

[0097] Table 4: Summary of elastin results

[0098]

[0099] Note: When performing statistical analysis, the significance of the NC group compared with the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##; the significance of the PC group and sample group compared with the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0100] like Figure 2Shown is a graph showing the changing trend of elastin content.

[0101] The results showed that compared with the BC group, the NC group fibroblasts received a total dose of 9J / cm 2 After long-wave ultraviolet radiation with melanin effect, the elastin content level decreased significantly (p-value<0.01), indicating that the radiation stimulation was effective.

[0102] Compared with the NC group, vitamin C and vitamin E in the PC group could significantly increase the elastin content level (p-value<0.01), indicating that the positive control test was effective.

[0103] Compared with the BC group, samples at concentrations of 1.25%, 2.5% and 5% (v / v) showed a significant improvement in the elastin content of fibroblasts induced by long-wave dark spot effect ultraviolet radiation (p-value<0.05), indicating that samples at all three concentrations have anti-wrinkle and firming effects.

[0104] 6. Conclusion

[0105] Under the test conditions, compared with the NC group, the sample recombinant human interleukin-2 (I) at concentrations of 1.25%, 2.5% and 5% (v / v) showed a significant improvement in the elastin content of fibroblasts induced by long-wave dark spot effect ultraviolet radiation (p-value<0.05), indicating that it has anti-wrinkle and firming effects.

[0106] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A use of recombinant human interleukin-2 (I) in the preparation of anti-wrinkle and firming products, characterized in that: Recombinant human interleukin 2 (I) is used as an active ingredient of the anti-wrinkle and firming product, wherein the volume concentration of the recombinant human interleukin 2 (I) is 1.25%-5%; The production method of recombinant human interleukin-2 (I) comprises the following steps: (1) Seed culture and fermentation: Take the recombinant human interleukin-2 (I) working seeds and inoculate them into the culture medium for culture. After the culture is completed, inoculate them into the fermentation tank for fermentation; (2) Collecting bacteria: remove the supernatant by centrifugation and collect the bacteria; (3) Preparation and dissolution of inclusion bodies: The bacterial suspension is broken with a high-pressure homogenizer, washed to remove some bacterial fragments and cell impurities, centrifuged to obtain the precipitate, and dissolved to obtain the inclusion body solution; (4) Crude extraction: The inclusion body solution is loaded and eluted to remove large molecular proteins and collect the target protein; (5) Desalting and renaturation: Collect the desalted protein solution, dilute it, add a catalyst, and oxidize and renature it to restore the protein's higher-order structure to obtain a renaturation solution; (6) Reverse high-performance liquid phase purification: The refolded liquid is further purified by a reversed-phase high-performance liquid phase system to remove impurities and obtain a high-purity sample solution; (7) Removing the mobile phase and filtering and sterilizing the stock solution: The sample solution collected by the HPLC is placed in a freeze dryer, and the organic solvent is removed by freeze drying. The sample is white and loose. The white and loose sample is dissolved with a buffer solution. After the solution is dissolved, it is the recombinant human interleukin-2 (I) stock solution; (8) Preparation of semi-finished products: Clean the liquid preparation and filtration system and two-stage filter cartridges, sterilize online with high-pressure steam, collect the excipients according to the amount indicated in the instruction sheet, dissolve them with injection water to make up the volume, slowly pour the recombinant human interleukin-2 (I) stock solution into the liquid preparation tank, stir and mix with the excipient solution, and sterilize and filter through a two-stage filter; (9) Packaging of finished products.

2. The use of a recombinant human interleukin-2 (I) according to claim 1 in the preparation of anti-wrinkle and firming products, characterized in that: The centrifugation operation in step (2) adopts a continuous flow centrifuge.

3. The use of a recombinant human interleukin-2 (I) according to claim 1 in the preparation of an anti-wrinkle and firming product, characterized in that: The elution operation in step (4) adopts an propylene dextran gel S-200 chromatography system.

4. The use of a recombinant human interleukin-2 (I) according to claim 1 in the preparation of an anti-wrinkle and firming product, characterized in that: In the step (5), the desalted protein solution is collected by loading the solution onto a Sephadex G25 chromatography system; the catalyst is cupric chloride.

5. The use of a recombinant human interleukin-2 (I) according to claim 1 in the preparation of an anti-wrinkle and firming product, characterized in that: The buffer solution in step (7) is sterilized and filtered using a filter membrane under a clean bench.

6. The use of a recombinant human interleukin-2 (I) according to claim 1 in the preparation of anti-wrinkle and firming products, characterized in that: The auxiliary materials in step (8) include phosphate, sodium lauryl sulfate, human albumin and mannitol.

7. The use of a recombinant human interleukin-2 (I) according to claim 1 in the preparation of an anti-wrinkle and firming product, characterized in that: The finished product packaging in step (9) includes: cleaning and sterilizing the rubber stopper, cleaning and sterilizing the vial, sterilizing the pot cover, taking out the sterilized canned parts from the sterilizer under the protection of Class A laminar flow, aseptically assembling them in sequence, starting normal canning after the filling volume is debugged and qualified, semi-pressing the stopper, and transferring the canned product to the freeze dryer through the transfer system for freeze drying. After the freeze drying is completed, the product is pressed and capped.

8. The use of recombinant human interleukin-2 (I) according to claim 1 in the preparation of anti-wrinkle and firming products, characterized in that: The anti-wrinkle and firming product comprises cosmetics. The recombinant human interleukin-2 (I) is added to a common carrier of cosmetics to prepare the anti-wrinkle and firming product. The common carrier is a combination of one or more of glycerol, vegetable oil, mineral oil, liposome, silicone oil and water.

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