Spray with moisturizing function and preparation method thereof

Abstract

The invention discloses spray with a moisturizing function and a preparation method thereof, and belongs to the technical field of daily chemical products. The natural biological moisturizing factor and the fibroblast growth factor are used, so that the moisturizing and skin barrier repairing effects are achieved, the resistance and the water retention capacity of the skin can be enhanced, the water balance of the skin is maintained, the moisturizing function is achieved, and water loss is prevented; meanwhile, synthesis of collagen and elastic fibers can be promoted, the elasticity and compactness of the skin can be improved, fine wrinkles and wrinkles are reduced, and the skin aging process is delayed, so that the skin is kept in a moist state for a long time, dryness and roughness are reduced, skin microcirculation is promoted, the blood supply and oxygen supply capacity of the skin is enhanced, and the skin can be effectively protected. Therefore, the overall health condition of the skin is improved; the moisturizing product is convenient to use and has a remarkable and continuous moisturizing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of daily chemicals, and specifically provides a spray with a moisturizing function and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body, protecting the various tissues and organs in the body from physical, chemical and pathogenic microorganisms. When the temperature drops or the body loses too much water, the stratum corneum of the skin cannot adjust enough moisturizing factors, the skin will become tight, and further discomfort will occur on the face. In order to keep the face moisturized, it is often necessary to apply moisturizing products from time to time, which is very troublesome and affects work.

[0003] The moisturizing effect provided by some existing moisturizing products is limited to short-term, such as the Chinese patent with publication number CN114617781A. Although it can keep the face moist in a short time because of the propellant contained in its composition, the face will return to a tight state after its composition evaporates quickly, and it is necessary to frequently reapply the product to maintain the effect. Some moisturizing products may cause skin allergies, irritation or other adverse reactions, such as the Chinese patent with publication number CN114081851A, which contains methylparaben in its composition and can cause skin allergies in some people. Therefore, the daily chemical field is in urgent need of a moisturizing product with a significant and continuous moisturizing effect that is easy to use.

[0004] In view of the above technical defects, a solution is now proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a spray with moisturizing effect and a preparation method thereof. The moisturizing spray provided by the present invention uses biological extracts as the main component and is supplemented with a small amount of auxiliary materials to effectively and permanently maintain facial moisture and has less irritation to the skin, will not cause excessive dryness of the skin, or even cause sensitivity or peeling.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a spray with moisturizing function is composed of the following components by weight: 10-15 parts of glycerin, 5-10 parts of butylene glycol, 2-5 parts of natural moisturizing factor (NMF), 0.5-3 parts of sodium hyaluronate, 2-5 parts of purslane extract, 2-5 parts of aloe water, 2-5 parts of collagen, 47-75 parts of purified water, and 1-2 mg / 0.1L of fibroblast growth factor (FGF).

[0007] Furthermore, the molecular weight of sodium hyaluronate is 6000-8000 Daltons, the purslane extract is a water-soluble purslane extract powder, and the specification is 15: 1. NMF is glutamine, serine and alanine, and the mass ratio thereof is 2: 1: 2.

[0008] Furthermore, the preparation method of FGF is divided into three steps: bacterial collection, protein extraction and protein purification;

[0009] Further, the bacterial cell collection method is as follows:

[0010] B1. Prepare broth culture medium. Weigh 25g broth culture medium powder and pour it into a 1L volumetric flask. Add 1L pure water and shake for 3min to fully dissolve the powder in the pure water. Sterilize it in an autoclave.

[0011] B2. Divide the sterilized broth culture medium into 5 sterile Erlenmeyer flasks, 200 ml in each bottle.

[0012] B3. Take out the FGF Escherichia coli culture medium, operate aseptically, and inoculate it into the broth culture medium in the triangular flask. The inoculation volume for each bottle is 20ul.

[0013] B4. Add antibiotics under sterile conditions. Choose ampicillin and add 20ul to each bottle.

[0014] B5. Seal the inoculated flask with tin foil, transfer to a shaker, and incubate for 24 hours.

[0015] B6. Collect the cultured E. coli in a 50 ml centrifuge tube, balance it, and centrifuge it at 8000 rpm and 4°C for 15 min. Pour off the supernatant and store it in a -80°C refrigerator.

[0016] Further, the protein extraction method is as follows:

[0017] All steps of this method were performed on ice;

[0018] C1. Add 0.5 ml of extraction reagent to 100 mg of wet weight of bacterial cells (add 1 ul of DTT and 10 ul of PMSF to each ml of extraction reagent before use) and repeatedly blow with a pipette tip to make it into a uniform suspension.

[0019] C2. Place the mixed bacterial solution in an ultrasonic disruptor and perform ultrasonic disruption for 20 minutes (the ultrasonic disruptor is set to 400W power, with 2s of ultrasonication and 10s of pause as one cycle) and repeat 2-3 times

[0020] C3. After the ultrasound is completed, centrifuge the bacterial solution at 12000 rpm and 4°C for 20 min to collect the supernatant.

[0021] Further, the protein purification method is as follows:

[0022] All steps of this method were performed on ice;

[0023] D1. Make NI 2+-NTA affinity chromatography column, slowly eluted with deionized water to avoid introducing bubbles into the column bed.

[0024] D2. Use 10 times the volume of column bed eluent for pre-equilibration, load the sample, and inject the supernatant after the bacteria are broken into NI 2+ -NTA affinity chromatography column, the bacterial solution slowly remains, repeated 2-3 times, and the target protein binds to the column bed.

[0025] D3. Rinse with 10 times the volume of the column bed with eluent, repeat 2-3 times to remove non-target protein impurities from the column bed.

[0026] D4. Use 5 times the column volume of an eluent containing 20 mM imidazole for elution, repeat 2-3 times, and collect the eluate. The target protein is present in the eluate collected this time.

[0027] The preparation method of the present invention is as follows:

[0028] A1: The preparation of fibroblast growth factor includes three steps: bacterial collection, protein extraction and protein purification;

[0029] A2: Add glycerol, butylene glycol and collagen into pure water and place on a magnetic stirrer, heat in a water bath, set the temperature to 65°C, set the speed to 1200 rpm, and stir for 30 minutes to obtain material A;

[0030] A3: Portulaca oleracea extract, aloe vera water and natural moisturizing factor (NMF) were mixed and dissolved in purified water, sodium hyaluronate and fibroblast growth factor (FGF) were added, and the mixture was placed on a magnetic stirrer, stirred at room temperature with the speed set to 600 rpm, and stirred for 30 min to obtain material B;

[0031] A4: Cool material A and material B to room temperature, and after cooling to room temperature, mix material A and material B and place them in a magnetic therapy mixer. Set the speed to 600 rpm and stir for 2 hours to obtain a spray with moisturizing function.

[0032] In summary, due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: the present invention can effectively neutralize free radicals, delay the skin aging process, reduce skin aging and damage caused by oxidation, and has a good moisturizing effect, can help the skin maintain water balance, soothe sensitive skin, accelerate the skin repair process, and has a certain therapeutic effect on damaged and inflamed skin. At the same time, it can promote the regeneration and renewal of skin cells, help improve the texture and gloss of the skin, and has certain antibacterial and antiviral activities, which can help remove bacteria and viruses on the skin surface, and help prevent and improve skin problems caused by microbial infection. The present invention can enhance the skin's resistance and water retention capacity, help maintain the skin's water balance and moisturizing function; can enhance the skin's barrier function, and help prevent water loss. It can stimulate the growth and regeneration of skin cells and accelerate the repair process of damaged skin tissue. It has a significant effect on the treatment of sunburn, trauma, or other injuries, so that the skin can recover to a healthy state faster, can promote the synthesis of collagen and elastic fibers, help improve the elasticity and firmness of the skin, reduce the appearance of fine lines and wrinkles, and delay the skin aging process; it helps to promote microcirculation and enhance the skin's blood supply and oxygen supply capacity, thereby improving the overall health of the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a flow chart of a method for preparing a spray with moisturizing function. DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and diagrams.

[0035] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0036] Example 1

[0037] In this example, E. coli BL21 (DE3) was used as the expression host; protein extraction used a protein extraction kit with the catalog number B5401048 produced by Shanghai Biotech; protein purification used a protein purification kit with the catalog number C601033 produced by Shanghai Biotech; broth culture medium powder used a broth culture medium powder with the catalog number A507002-0250 produced by Shanghai Biotech; ampicillin powder with the catalog number A102050 purchased from Aladdin was used; steps 7-13 were performed on ice.

[0038] 1. Prepare broth culture medium. Weigh 25g broth culture medium powder and pour it into a 1L volumetric flask. Add 1L pure water and shake for 3min to fully dissolve the powder in the pure water. Sterilize it in an autoclave.

[0039] 2. Divide the sterilized broth culture medium into 5 sterile Erlenmeyer flasks, 200 ml in each bottle.

[0040] 3. Take out the FGF Escherichia coli culture medium, aseptically operate, and inoculate it into the broth culture medium in the triangular flask, with an inoculation volume of 20ul per bottle.

[0041] 4. Add antibiotics under sterile conditions. Choose ampicillin and add 20ul to each bottle.

[0042] 5. Seal the inoculated flask with tin foil, transfer to a shaker, and culture for 24 hours.

[0043] 6. Use a 50ml centrifuge tube to collect the cultured E. coli, balance it, and centrifuge it at 8000rpm and 4℃ for 15min. Pour off the supernatant and get 4 tubes of 2.5g of bacteria. One tube of bacteria is used for the subsequent process and the remaining bacteria are stored in a -80℃ refrigerator.

[0044] 7. Add 12.5 ml of extraction reagent to a tube of 2.5 g of wet weight bacteria (add 1 ul of DTT and 10 ul of PMSF to each ml of extraction reagent before use) and repeatedly blow with a pipette tip to make it into a uniform suspension.

[0045] 8. Place the mixed bacterial solution in an ultrasonic disruptor and perform ultrasonic disruption for 20 minutes (the ultrasonic disruptor is set to 400W power, and ultrasonication for 2S and pause for 10S as a cycle) and repeat 2-3 times.

[0046] 9. After the ultrasound is completed, centrifuge the bacterial solution at 12000rpm and 4℃ for 20min to collect the supernatant.

[0047] 10. Record the NI 2+ -NTA affinity chromatography column, slowly eluted with deionized water to avoid introducing bubbles into the column bed.

[0048] 11. Use 15 ml of eluent for pre-balance, load the sample, and inject the supernatant after the bacteria are broken into NI 2+ -NTA affinity chromatography column, the bacterial solution slowly remains, repeated 3 times, and the target protein binds to the column bed.

[0049] 12. Rinse with 15 ml of eluent and repeat 3 times to remove non-target protein impurities on the column bed.

[0050] 13. Use 7.5 ml of elution solution containing 20 mM imidazole for elution, repeat 3 times, collect the eluate, and obtain a total of 7.5 ml of eluate containing FGF.

[0051] The protein content of the eluate was determined using the CBB method of the protein assay kit (C035-2-1, Nanjing Jiancheng)

[0052] Table 1, CBB method determination system

[0053]

[0054] The FGF protein content in the eluate was determined to be 681.2 ug / ml.

[0055] Example 2

[0056] 1. Add 10 ml of glycerol, 5 ml of butylene glycol and 2 g of collagen to 75 ml of pure water and place it on a magnetic stirrer. Heat in a water bath with the temperature set to 65°C and the speed set to 1200 rpm. Stir for 30 min to obtain material A.

[0057] 2. Mix and dissolve 2 g of purslane extract, 2 ml of aloe water and 2 g of natural moisturizing factor (NMF) in pure water, add 0.5 g of sodium hyaluronate and 1.6 ml of the eluent prepared in Example 1 (containing 1 mg of FGF), place the mixture on a magnetic stirrer, stir at room temperature, set the speed to 600 rpm, and stir for 30 min to obtain material B.

[0058] 3. Cool material A and material B to room temperature, and after cooling to room temperature, mix material A and material B and place them in a magnetic therapy stirrer, set the speed to 600 rpm and stir for 2 hours to obtain this embodiment.

[0059] Table 2, reagent parameters used in Example 2

[0060] Drug Name source model parameter glycerin Nanjing Rongji Chemical Co., Ltd. KG-250 250kg Butanediol Hunan Fuqian Pharmaceutical Co., Ltd. 107-88-0 25kg Collagen Hainan Huayan Biotechnology Co., Ltd. HYCTP30-J 500g Purslane Extract Xi'an Jinheng Chemical Co., Ltd. JH01 10:1 Aloe Vera Water Guangzhou Baiyu Biotechnology Co., Ltd. 2019 10％ Sodium Hyaluronate Xi'an Zhiyuan Biotechnology Co., Ltd. 9067-32-7 1kg NMF Guangzhou Zhongguang Biotechnology Co., Ltd. 107-43-7 1kg

[0061] Example 3

[0062] 1. Add 15 ml of glycerol, 10 ml of butylene glycol and 5 g of collagen to pure water and place it on a magnetic stirrer. Heat it in a water bath with the temperature set to 65°C and the speed set to 1200 rpm. Stir for 30 minutes to obtain material A.

[0063] 2. Mix and dissolve 5 g of purslane extract, 5 ml of aloe water and 5 g of natural moisturizing factor (NMF) in pure water, add 3 g of sodium hyaluronate and 3.2 ml of the eluent prepared in Example 1 (containing 2 mg of FGF), place the mixture on a magnetic stirrer, stir at room temperature, set the speed to 600 rpm, and stir for 30 min to obtain material B.

[0064] 3. Cool material A and material B to room temperature, and after cooling to room temperature, mix material A and material B and place them in a magnetic therapy stirrer, set the speed to 600 rpm and stir for 2 hours to obtain this embodiment.

[0065] Table 3, reagent parameters used in Example 3

[0066]

[0067]

[0068] Comparative Example 1

[0069] Example 1 of Chinese patent with publication number CN114081851A is used as comparative example 1

[0070] Comparative Example 2

[0071] Example 1 of Chinese patent with publication number CN109568568A is used as comparative example 2

[0072] Cytotoxicity assay:

[0073] Example 2, Example 3, Comparative Example 1 and Comparative Example 2 were diluted to 1% and 10% respectively using sterile water to prepare concentration gradients for cytotoxicity testing.

[0074] 1. A cytotoxicity experiment was performed using 293T cells and an MTT kit with a catalog number of M1020 produced by Solarbio to detect the toxic effect of the spray with moisturizing function prepared in Examples 2 and 3 on cells.

[0075] 2. The MTT kit used contains MTT solution and Formazan solution.

[0076] 3. Resuscitate and culture 293T cells. Collect logarithmic phase 293T cells one day later, adjust the concentration of cell suspension, and distribute them in 96-well plates, 180ul per well, 3000-10000 cells / well.

[0077] 4. Place the 96-well plate at 37°C, 5% CO 2 Incubate in a constant temperature incubator for 6 hours.

[0078] 5. Add 10ul of the test sample to each well and set up 3 groups for each gradient.

[0079] 6. After aspirating the supernatant, add 90ul of fresh culture medium, then add 10ul of MTT solution and continue culturing for 4h.

[0080] 7. Then aspirate the supernatant, add 110ul Formazan solution to each well, place on a shaker and shake for 10 minutes to fully dissolve the crystals. After shaking, transfer the 96-well plate to an ELISA reader and measure the absorbance of each well at 490nm.

[0081] 8. Set up blank wells (culture medium, MTT solution, Formazan solution) and control wells (293T cells, sterile water, culture medium, MTT solution, Formazan solution) at the same time, with 3 replicates per well.

[0082] Table 4, MTT cytotoxicity test results

[0083] Grouping Cytotoxicity Cell viability 1% Example 2 Non-toxic / 1% Example 3 Non-toxic / 1%Comparative Example 1 Non-toxic / 1%Comparative Example 2 Non-toxic / 10% Example 2 Non-toxic / 10% Example 3 Non-toxic / 10% Comparative Example 1 Non-toxic / 10% Comparative Example 2 Non-toxic / 100% Example 2 2.21％ 97.79％ 100% Example 3 1.97％ 98.03％ 100% Comparative Example 1 7.37％ 92.63％ 100% Comparative Example 2 8.04％ 91.96％

[0084] It can be seen from Table 1 that Example 2 and Example 3 have no cytotoxicity when the dilution ratio is 10%.

[0085] Effect detection:

[0086] Twenty volunteers with similar facial dryness were recruited and divided into five groups of four people in each group. Each group was sprayed with Example 2 (dilution ratio 10%), Example 3 (dilution ratio 10%), Comparative Example 1 (dilution ratio 10%), Comparative Example 2 (dilution ratio 10%) and clean water every two hours starting from 8 a.m. The facial moisture value was tested every hour using German CK (MKA9). The test was continued for 8 hours, and the average value was taken for each group to calculate the moisture change value within 8 hours.

[0087] Table 5, Skin moisture values ​​of test groups

[0088] Group initial 0h 1h 2h 3h 4h 5h 6h 7h Example 2 55 87 72 94 79 101 86 112 98 Example 3 53 89 75 97 82 105 90 118 100 Comparative Example 1 57 85 68 91 71 98 78 109 94 Comparative Example 2 55 85 70 90 74 95 81 108 96 Purified water 54 82 57 86 57 84 56 86 57

[0089] Through observation, the faces of the volunteers sprayed with Example 2 and Example 3 were obviously more moisturized and smooth. From the experimental data, the moisture change values ​​of Example 2 and Example 3 were higher than those of Comparative Example 1 and Comparative Example 2 from 1 hour onwards. The moisture of the face of the volunteers sprayed with pure water basically returned to the initial value after one hour, which shows that the moisturizing spray used in Example 2 and Example 3 does have a continuous moisturizing effect, and the face is more moisturized and smooth after use; among them, the moisturizing spray prepared in Example 3 has a more excellent effect.

[0090] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A spray with moisturizing function, characterized in that: The composition is composed of the following components by weight: 10-15 parts of glycerin, 5-10 parts of butylene glycol, 2-5 parts of natural moisturizing factor, 0.5-3 parts of sodium hyaluronate, 2-5 parts of purslane extract, 2-5 parts of aloe vera water, 2-5 parts of collagen, 47-75 parts of purified water and 1-2 mg / 0.1L of fibroblast growth factor; The fibroblast growth factor is obtained by collecting transgenic Escherichia coli through bacterial culture, protein extraction and protein purification.

2. The method for preparing a spray with moisturizing function according to claim 1, characterized in that: The following steps are involved: A1: The preparation of fibroblast growth factor includes three steps: bacterial collection, protein extraction and protein purification; A2: Add glycerol, butylene glycol and collagen into pure water and place on a magnetic stirrer, heat in a water bath, set the temperature to 65°C, set the speed to 1200 rpm, and stir for 30 minutes to obtain material A; A3: Mix and dissolve the purslane extract, aloe vera water and natural moisturizing factor in pure water, add sodium hyaluronate and fibroblast growth factor, place the mixture on a magnetic stirrer, stir at room temperature, set the speed to 600 rpm, and stir for 30 minutes to obtain material B; A4: Cool material A and material B to room temperature, and after cooling to room temperature, mix material A and material B and place them in a magnetic therapy mixer. Set the speed to 600 rpm and stir for 2 hours to obtain a spray with moisturizing function.

3. The method for preparing a spray with moisturizing function according to claim 2, characterized in that: The bacterial cell collection comprises the following steps: B1: Prepare broth medium. Weigh 25g broth medium powder into a 1L volumetric flask. Add 1L pure water and shake for 3min to fully dissolve the powder in the pure water. Sterilize in an autoclave. B2: The sterilized broth medium is divided into 5 sterile Erlenmeyer flasks, 200 ml per bottle; B3: Take out the FGF E. coli culture medium, operate aseptically, and inoculate it into the broth medium in the triangular flask, with an inoculation volume of 20ul per bottle; B4: Add antibiotics aseptically, choose ampicillin, and add 20ul to each bottle; B5: Seal the inoculated flask with tin foil, transfer to a shaker, and incubate for 24 hours; B6: Use a 50ml centrifuge tube to collect the cultured E. coli, balance it, centrifuge it at 8000rpm, 4℃ for 15min, pour off the supernatant, and store it in a -80℃ refrigerator.

4. The method for preparing a spray with moisturizing function according to claim 2, characterized in that: The protein extraction comprises the following steps: All steps of this method were performed on ice; C1: Add 0.5 ml of extraction reagent to 100 mg of wet weight of bacterial cells. Before use, add 1 ul of DTT and 10 ul of PMSF to each ml of extraction reagent and repeatedly blow with a pipette tip to make it a uniform suspension; C2: Place the mixed bacterial solution in an ultrasonic disruptor and perform ultrasonic disruption for 20 minutes. Set the parameters of the ultrasonic disruptor to 400W power, and repeat the ultrasonication cycle 2S and 10S for 2-3 times. C3: After the ultrasound is completed, the bacterial liquid is centrifuged at 12000 rpm and 4°C for 20 min to collect the supernatant.

5. The method for preparing a spray with moisturizing function according to claim 2, characterized in that: The protein purification The following steps are involved: D1: Fill the NI2+-NTA affinity chromatography column and slowly elute it with deionized water to avoid introducing bubbles on the column bed; D2: Use 10 times the volume of column bed eluent for pre-equilibration, load the sample, and inject the supernatant after the bacteria are broken into NI 2+ -In the NTA affinity chromatography column, the bacterial solution flows slowly, and this process is repeated 2-3 times until the target protein binds to the column bed; D3: Rinse with 10 times the volume of the column bed eluent, repeat 2-3 times to remove non-target protein impurities from the column bed; D4: Use 5 times the column volume of an eluent containing 20 mM imidazole for elution, repeat 2-3 times, collect the eluent, and obtain an eluent containing fibroblast growth factor.

6. The spray with moisturizing function according to claim 1, characterized in that: The natural moisturizing factors are glutamine, serine and alanine, wherein the mass ratio of glutamine, serine and alanine is 2:1:

2.

7. The spray with moisturizing function according to claim 1, characterized in that: The molecular weight of the sodium hyaluronate is 6000-8000 Daltons.

8. The spray with moisturizing function according to claim 1, characterized in that: The purslane extract is water-soluble purslane extract powder with a specification of 15:1.

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